The protocol presents a method for isolating whole cell protein lysates from dissected mouse embryo facial processes or cultured mouse embryonic palatal mesenchyme cells and performing subsequent western blotting to assess phosphorylated protein levels.
Mammalian craniofacial development is a complex morphological process during which multiple cell populations coordinate to generate the frontonasal skeleton. These morphological changes are initiated and sustained through diverse signaling interactions, which often include protein phosphorylation by kinases. Here, two examples of physiologically-relevant contexts in which to study phosphorylation of proteins during mammalian craniofacial development are provided: mouse facial processes, in particular E11.5 maxillary processes, and cultured mouse embryonic palatal mesenchyme cells derived from E13.5 secondary palatal shelves. To overcome the common barrier of dephosphorylation during protein isolation, adaptations and modifications to standard laboratory methods that allow for isolation of phosphoproteins are discussed. Additionally, best practices are provided for proper analysis and quantification of phosphoproteins following western blotting of whole cell protein lysates. These techniques, particularly in combination with pharmacological inhibitors and/or murine genetic models, can be used to gain greater insight into the dynamics and roles of various phosphoproteins active during craniofacial development.
Mammalian craniofacial development is a complex morphological process during which multiple cell populations coordinate to generate the frontonasal skeleton. In the mouse, this process begins at embryonic day (E) 9.5 with the formation of the frontonasal prominence and pairs of maxillary and mandibular processes, each of which contains post-migratory cranial neural crest cells. The lateral and medial nasal processes arise from the frontonasal prominence with the appearance of the nasal pits and eventually fuse to form the nostrils. Further, the medial nasal processes and maxillary processes fuse to generate the upper lip. Concurrently, palatogenesis is initiated with the formation of distinct outgrowths – the secondary palatal shelves – from the oral side of the maxillary processes at E11.5. Over time, the palatal shelves grow downward on either side of the tongue, elevate to an opposing position above the tongue, and eventually fuse at the midline to form a continuous palate that separates the nasal and oral cavities by E16.51.
These morphological changes throughout craniofacial development are initiated and sustained through diverse signaling interactions, which often include protein phosphorylation by kinases. For example, cell membrane receptors, such as subfamilies of transforming growth factor (TGF)-β receptors, including bone morphogenetic protein receptors (BMPRs), and various receptor tyrosine kinase (RTK) families, are autophosphorylated upon ligand binding and activation in cranial neural crest cells2,3,4. Additionally, the G protein-coupled transmembrane receptor Smoothened becomes phosphorylated in cranial neural crest cells and craniofacial ectoderm downstream of Sonic hedgehog (SHH) ligand binding to the Patched1 receptor, resulting in Smoothened accumulation at the ciliary membrane and SHH pathway activation5. Such ligand-receptor interactions can occur through autocrine, paracrine, and/or juxtacrine signaling in craniofacial contexts. For example, BMP6 is known to signal in an autocrine manner during chondrocyte differentiation6, whereas fibroblast growth factor (FGF) 8 is expressed in the pharyngeal arch ectoderm and binds to members of the FGF family of RTKs expressed in the pharyngeal arch mesenchyme in a paracrine fashion to initiate patterning and outgrowth of the pharyngeal arches7,8,9,10. Furthermore, Notch signaling is activated in both chondrocytes and osteoblasts during craniofacial skeletal development through juxtacrine signaling when transmembrane Delta and/or Jagged ligands bind to transmembrane Notch receptors on neighboring cells, which are subsequently cleaved and phosphorylated11. However, there are other ligand and receptor pairs important for craniofacial development that have the flexibility to function in both autocrine and paracrine signaling. As an example, during murine tooth morphogenesis, platelet-derived growth factor (PDGF)-AA ligand has been demonstrated to signal in an autocrine manner to activate the RTK PDGFRα in the enamel organ epithelium12. In contrast, in murine facial processes during mid-gestation, transcripts encoding the ligands PDGF-AA and PDGF-CC are expressed in the craniofacial ectoderm, while the PDGFRα receptor is expressed in the underlying cranial neural crest-derived mesenchyme, resulting in paracrine signaling13,14,15,16,17. Regardless of the signaling mechanism, these receptor phosphorylation events often result in the recruitment of adaptor proteins and/or signaling molecules, which frequently become phosphorylated themselves to initiate intracellular kinase cascades such as the mitogen-activated protein kinase (MAPK) pathway18,19.
The terminal intracellular effectors of these cascades can then phosphorylate an array of substrates, such as transcription factors, RNA-binding, cytoskeletal and extracellular matrix proteins. Runx220, Hand121, Dlx3/522,23,24, Gli1-325, and Sox926 are among the transcription factors phosphorylated in the context of craniofacial development. This post-translational modification (PTM) can directly affect susceptibility to alternative PTMs, dimerization, stability, cleavage, and/or DNA-binding affinity, among other activities20,21,25,26. Additionally, the RNA-binding protein Srsf3 is phosphorylated in the context of craniofacial development, leading to its nuclear translocation27. In general, phosphorylation of RNA-binding proteins has been shown to affect their subcellular localization, protein-protein interactions, RNA binding, and/or sequence specificity28. Furthermore, phosphorylation of actomyosin can lead to cytoskeletal rearrangements throughout craniofacial development29,30, and phosphorylation of extracellular matrix proteins, such as small integrin-binding ligand N-linked glycoproteins, contributes to biomineralization during skeletal development31. Through the above and numerous other examples, it is evident that there are wide implications for protein phosphorylation during craniofacial development. Adding an additional level of regulation, protein phosphorylation is further modulated by phosphatases, which counteract kinases by removing phosphate groups.
These phosphorylation events at both the receptor and effector molecule levels are critical for the propagation of signaling pathways and ultimately result in changes in gene expression in the nucleus, driving specific cell activities, such as migration, proliferation, survival, and differentiation, which result in proper formation of the mammalian face. Given the context specificity of protein interactions with kinases and phosphatases, the resulting changes in PTMs, and their effects on cell activity, it is critical that these parameters be studied in a physiologically-relevant setting to gain complete understanding of the contribution of phosphorylation events to craniofacial development. Here, examples of two contexts in which to study phosphorylation of proteins and, thus, activation of signaling pathways during mammalian craniofacial development are provided: mouse facial processes, in particular E11.5 maxillary processes, and cultured mouse embryonic palatal mesenchyme cells derived from E13.5 secondary palatal shelves – both primary32 and immortalized33. At E11.5, the maxillary processes are in the process of fusing with the lateral and medial nasal processes1, thereby representing a critical timepoint during mouse craniofacial development. Further, maxillary processes and cells derived from the palatal shelves were chosen here because the latter structures are derivatives of the former, thereby providing researchers the opportunity to interrogate protein phosphorylation in vivo and in vitro in related contexts. However, this protocol is also applicable to alternative facial processes and developmental timepoints.
A critical problem in studying phosphorylated proteins is that they are easily dephosphorylated during protein isolation by abundant environmental phosphatases. To overcome this barrier, adaptations and modifications to standard laboratory methods that allow for isolation of phosphorylated proteins are discussed. Additionally, best practices are provided for proper analysis and quantification of phosphorylated proteins. These techniques, particularly in combination with pharmacological inhibitors and/or murine genetic models, can be used to gain greater insight into the dynamics and roles of various signaling pathways active during craniofacial development.
All the procedures involving animals were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Colorado Anschutz Medical Campus and performed in compliance with institutional guidelines and regulations. Female 129S4 mice at 1.5-6 months of age and housed at a sub-thermoneutral temperature of 21-23 °C were used for embryo harvests. A schematic workflow of the protocol is represented in Figure 1. See the Table of Materials for details regarding all materials, equipment, software, reagents, and animals used in this protocol.
1. Harvesting E11.5 mouse embryos
2. Dissecting maxillary processes from E11.5 mouse embryos
3. Isolating whole cell protein lysates from mouse maxillary processes
4. Isolating whole cell protein lysates from primary and/or immortalized mouse embryonic palatal mesenchyme (MEPM) cells
NOTE: Isolation and culture of primary MEPM cells from E13.5 mouse embryos and immortalized MEPM cells have been previously described32,33,34. Stimulation of cells with growth factor (steps 4.1-4.7) can be performed prior to cell lysis. If non-stimulated cells are desired, proceed to step 4.8 below.
5. Western blotting of whole cell protein lysates from mouse facial processes and/or MEPM cells for phosphoproteins
When attempting to characterize the phosphorylation of proteins isolated from mouse facial processes and/or cultured palatal mesenchyme cells, the representative results will ideally reveal a distinct, reproducible band following western blotting with an anti-phosphoprotein antibody that runs at or near the height of the corresponding total protein band (Figure 3). However, if extensive phosphorylation of the protein occurs, there may be a slight upward shift of the phosphoprotein band compared to that of the total protein band. Further, if multiple isoforms of a protein exist in a tissue, each of which is phosphorylated, or the protein is variably subjected to additional PTMs, multiple bands may appear in the western blot with an anti-phosphoprotein antibody. If no signal is observed for the phosphoprotein of interest, despite the detection of the total protein of interest, the protein may not be phosphorylated in the chosen context, or a technical issue may have arisen with the protocol. In the latter case, western blotting of whole cell lysates can be performed with anti-phosphoserine/threonine and/or anti-phosphotyrosine antibodies to indicate whether the protocol worked as expected. As phosphorylation of proteins is abundant in craniofacial tissues25,37, the presence of multiple phosphoprotein bands will indicate successful completion of the protocol and accurate results from the initial screening.
It is often useful to compare phosphoprotein levels between wild-type and mutant embryo tissues or in response to the treatment of cultured cells. In the former case, a difference in phosphoprotein levels between genotypes would appear as a difference in band intensities for the phosphoprotein of interest with similar levels of the total protein of interest27. In the latter case, representative outcomes would reveal increased phosphoprotein band intensities compared to those of untreated cells upon treatment with, for example, a growth factor (Figure 3) and decreased band intensities following treatment with a kinase inhibitor27,37. For quantitation of these differences, levels of the phosphoprotein of interest would be normalized to levels of the total protein of interest. The levels of the total protein of interest are expected be relatively equal between samples, a finding which should be confirmed by separate western blotting using one or more antibodies against loading and expression control proteins such as β-tubulin, β-actin, and/or GAPDH (Figure 3). For growth factor treatment of cultured cells, positive results will likely show an increase in phosphoprotein levels in a relatively short time frame of 2-15 min following stimulation (Figure 3). There should be little or no phosphorylation in the absence of growth factor treatment, unless there are other factors at play resulting in phosphorylation of the protein, examples of which include endogenous expression of growth factors by the cells, phosphorylation of the protein by another signaling molecule expressed by the cells, and/or a mutation that leads to constitutive phosphorylation of the protein in the absence of growth factor treatment. These factors should be considered in the interpretation of the results. Negative results include no change in phosphorylation in response to a timecourse of growth factor treatment, in which case the protocol should be evaluated for maintenance of phosphoproteins as detailed above. Additionally, the process of membrane stripping can occasionally affect, and reduce, total protein levels. In this case, opposite trends for the amount of the phosphoprotein of interest versus the total protein of interest would be observed. This is suboptimal, as total protein levels are expected to be relatively equal across samples, assuming equal loading and expression of the protein, with changes occurring only in phosphoprotein levels.
Figure 1: Workflow for isolation of whole cell protein lysates from mouse facial processes and cultured palatal mesenchyme cells for phosphoprotein analysis. Maxillary processes are dissected from E11.5 mouse embryos on ice, and/or the medium is aspirated from cultured MEPM cells at 80%-100% confluence, which are subsequently washed twice with ice-cold PBS. Whole cell protein lysates are isolated using lysis buffer with protease and phosphatase inhibitors, followed by SDS-PAGE, electrotransfer to a PVDF membrane, blocking of the membrane with BSA, probing for the phosphoprotein, stripping of the membrane, reprobing for total protein, and quantitation of band intensities. Abbreviations: LNP = lateral nasal process; MxP = maxillary process; MdP = mandibular process; PA2 = second pharyngeal arch; PBS = phosphate-buffered saline; FBS = fetal bovine serum; PS = palatal shelf; MEPM = mouse embryonic palatal mesenchyme; SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PVDF = polyvinylidene fluoride; p-P = phosphorylated protein. Please click here to view a larger version of this figure.
Figure 2: Dissection of maxillary processes. (A) Lateral view of an E11.5 mouse embryo with the three cuts (labeled 1-3) required to separate the MxP from the face indicated by colored lines. (B) Lateral view of an E11.5 mouse embryo following removal of MxP outlined with a dashed black line. (C) Dissected MxP. (D) A 35 mm Petri dish set-up with small droplets of PBS, 2% trypsin, and 10% FBS for optional separation of MxP ectoderm and mesenchyme. (E) MxP with Ect partially separated from Mes. (F) Separated MxP Ect and Mes. Scale bars: 1 mm (A–C,E,F), 10 mm (D). Abbreviations: LNP = lateral nasal process; MxP = maxillary process; MdP = mandibular process; PA2 = second pharyngeal arch; PBS = phosphate-buffered saline; FBS = fetal bovine serum; Mes = mesenchyme; Ect = ectoderm. Please click here to view a larger version of this figure.
Figure 3: PDGFRβ and MAPK effector Erk1/2 phosphorylation in response to PDGF-BB ligand treatment of immortalized MEPM cells. Western blot analysis of whole cell lysates from immortalized MEPM cells following a timecourse of 10 ng/mL PDGF-BB ligand stimulation from 2 min to 15 min with anti-phospho-PDGFR, anti-PDGFRβ, anti-phospho-Erk1/2, anti-Erk1/2, and anti-β-tubulin (E7) antibodies. Multiple bands in anti-PDGFRβ blot represent differentially glycosylated proteins. Abbreviations: PDGF = platelet-derived growth factor; WCL = whole cell lysate; kD = kilodalton; WB = western blot; p-PDGFR = phosphorylated PDGFR; PDGFR = PDGF receptor; p-Erk = phosphorylated Erk; Erk = extracellular signal-regulated kinase. Please click here to view a larger version of this figure.
The protocol described here allows researchers to probe critical phosphorylation-dependent signaling events during craniofacial development in a robust and reproducible manner. There are several critical steps in this protocol that ensure proper collection of data and analysis of results. Whether isolating phosphoproteins from mouse facial processes and/or cultured palatal mesenchyme cells, it is imperative to move quickly and efficiently while keeping all reagents and materials on ice when indicated. The low temperature of ice slows down metabolic activity in the cells, thereby protecting phosphorylated proteins from phosphatase activity38 and maintaining a high protein yield. The use of three separate 10 cm Petri dishes during the dissection of maxillary processes ensures that all dissections take place in sufficiently prechilled histology PBS. Relatedly, an additional, essential component of this protein isolation protocol is the use of phosphatase inhibitors in all lysis buffers. These reagents also protect phosphate groups on the protein of interest39,40 and maintain the integrity of the phosphorylated protein for further analysis by western blotting.
This protocol additionally introduces modifications that allow for more specific analyses of phosphoproteins in tissue and cultured cells. In the process of dissecting facial processes, optional steps have been added for the separation of the maxillary process ectoderm and mesenchyme to allow for the study of compartment-specific protein phosphorylation27. To test for efficient isolation of these tissue layers, users can assess the expression of genes enriched in the ectoderm, such as Gabrp, Trim29, Esrp1, and Mpzl2, and/or genes enriched in the mesenchyme, such as Aldh1a2 and AW55198441. For the isolation of phosphoproteins from cultured palatal mesenchyme cells, optional steps have been added for growth factor stimulation. In this case, cells should be serum starved in medium containing 0.0%-0.1% serum. Standard serums such as FBS contain numerous growth factors42 that can convolute results from exogenous growth factor stimulation. Thus, temporary serum starvation primes cells to respond to acute growth factor treatment.
This protocol also provides optimized methods to accurately quantify protein phosphorylation. In the western blotting steps, it is important to block the PVDF membrane using BSA rather than milk, which is used in many standard western blotting techniques. This switch is imperative, as milk contains the phosphoprotein casein43 and leads to high background signal in the analysis of other phosphoproteins. Further, it is important that levels of the phosphorylated protein of interest are quantified against levels of the total protein of interest rather than levels of a standard loading control protein. This way, any changes in the expression of the total protein of interest can be accounted for in the quantification of phosphoprotein levels. If protein yield in general is too low for accurate phosphoprotein detection, tissue samples from the same stage and genotype may be pooled27, and/or cells can be grown in larger cell culture vessels than those described here for future experiments. Alternatively, if stripping consistently appears to alter the total protein levels, two approaches can be taken to modify the described protocol to achieve reproducible results: 1) use of an antibody scheme that employs anti-phosphoprotein and anti-protein antibodies generated in different host species combined with two species-specific secondary antibodies and separate (if using HRP-conjugated secondary antibodies) or simultaneous (if using fluorophore-conjugated secondary antibodies) development of the blots; or 2) simultaneous processing of two identical membranes – the first incubated with an anti-phosphoprotein antibody, the second with an anti-protein antibody. In both alternative approaches, the levels of the phosphorylated protein of interest should still be quantified against the levels of the total protein of interest. Finally, as phosphorylation is a dynamic process, phosphoprotein levels should be assessed in at least three biological replicates per condition. Mouse facial process tissue or primary MEPM cells derived from an individual embryo or an individual pool of embryos from the same stage and genotype would constitute a single biological replicate. Similarly, immortalized MEPM cells of different passages would constitute biological replicates. If cultured cells are treated with growth factor, treatment of biological replicates should occur on different days using separate aliquots of growth factor.
Two limitations of the approach presented here exist, which are discussed below and should be considered before initiating the protocol. The first involves the dynamic range of ECL. The ECL western blotting substrate recommended in this protocol has a wide dynamic range, with a low picogram sensitivity. However, empirically, proteins of low abundance or low phosphorylation status may be difficult to detect using these methods. If phosphorylated proteins are undetectable following 20 min of ECL substrate incubation, it is recommended to wash the membrane in TBS-T as described and instead incubate the membrane in a high-sensitivity ECL western blotting substrate with a low femtogram sensitivity. Alternatively, fluorophore-conjugated secondary antibodies can be used, which offer a wider dynamic range than enzyme-based approaches such as ECL44. A second limitation arises from the normalization procedure. As described extensively, it is recommended that signal from the phosphoprotein of interest be normalized to the signal from the total protein of interest. However, this process relies on the assumption that the levels of the total protein of interest do not change between samples, which is often confirmed by separate western blotting using antibodies against one or more housekeeping proteins. An important consideration though is that the levels of housekeeping proteins may change between samples due to uneven loading, incomplete transfer to the membrane, and/or differences in protein expression. Thus, to enhance accuracy during normalization, researchers can consider moving to implement a true total protein stain, such as Ponceau S45, which detects all proteins in each lane of the membrane and accounts for these technical limitations.
Overall, this protocol overcomes the problem of protein dephosphorylation that commonly occurs during protein isolation. Accurate analysis of protein phosphorylation will help researchers gain a more precise understanding of the role of phosphorylated proteins during craniofacial development. Furthermore, this protocol offers robust and efficient methods to analyze phosphorylated protein levels in physiologically-relevant contexts, each with their own benefits. While protein lysates from embryo tissues provide a static snapshot of protein phosphorylation in vivo, the implementation of cultured cells supplies data on dynamic phosphorylation responses to acute treatments. This protocol has the capacity to directly confirm and expand upon results and hypotheses stemming from large datasets, e.g., mass spectrometry37 and RNA-sequencing27, to discover new interactions within signaling networks. Importantly, this protocol can be used to probe how perturbations in phosphorylation directly influence intracellular signaling, gene expression, and cellular activity in craniofacial contexts. These diverse applications will greatly enhance understanding of the effects of protein phosphorylation in craniofacial development.
The authors have nothing to disclose.
129S4 mice were a gift from Dr. Philippe Soriano, Icahn School of Medicine at Mount Sinai. This work was supported with funds from the National Institutes of Health (NIH)/National Institute of Dental and Craniofacial Research (NIDCR) R01 DE027689 and K02 DE028572 to K.A.F., F31 DE029976 to M.A.R. and F31 DE029364 to B.J.C.D.
Equipment | |||
Block for mini dry bath | Research Products International Corp | 400783 | |
ChemiDoc XRS+ imaging system with Image Lab software | Bio-Rad | 1708265 | chemiluminescence imager |
CO2 incubator, air jacket | VWR | 10810-902 | |
Dissecting board, 11 x 13 in | Fisher Scientific | 09 002 12 | |
Electrophoresis cell, 4-gel, for mini precast gels with mini trans-blot module | Bio-Rad | 1658030 | |
Hybridization oven | Fisher Scientific | UVP95003001 | |
Microcentrifuge 5415 D with F45-24-11 rotor (Eppendorf) | Sigma Aldrich | Z604062 | |
Mini dry bath | Research Products International Corp | 400780 | |
Orbital shaker | VWR | 89032-092 | |
pH meter | VWR | 89231-662 | |
Power supply for SDS-PAGE | Bio-Rad | 1645050 | |
Rectangular ice pan, maxi 9 L | Fisher Scientific | 07-210-093 | |
Stemi 508 stereo microscope with stand K LAB, LED ring light | Zeiss | 4350649020000000 | dissecting microscope |
Timer | VWR | 62344-641 | |
Tube revolver | Fisher Scientific | 11 676 341 | |
Vortex mixer | Fisher Scientific | 02 215 414 | |
Water bath | VWR | 89501-472 | |
Western blot box | Fisher Scientific | NC9358182 | |
Materials | |||
Cell culture dishes, 6 cm | Fisher Scientific | 12-565-95 | |
Cell culture plates, 12 well | Fisher Scientific | 07-200-82 | |
Cell lifters | Fisher Scientific | 08-100-240 | |
CO2 | Airgas | CD USP50 | |
Conical tubes, polypropylene, 50 mL | Fisher Scientific | 05-539-13 | |
Dumont #5 fine forceps | Fine Science Tools | 11254-20 | |
Embryo spoon | Fine Science Tools | 10370-17 | |
Microcentrifuge tubes, 0.5 mL | VWR | 89000-010 | |
Microcentrifuge tubes, 1.5 mL | VWR | 20170-038 | |
Pasteur pipet, 5.75" | Fisher Scientific | 13-678-6A | |
Pasteur pipet, 9" | VWR | 14672-380 | |
Petri dishes, 10 cm | Fisher Scientific | 08-757-100D | |
Petri dishes, 35 mm | Fisher Scientific | FB0875711YZ | |
Pouches, transparent, polyethylene lining | Fisher Scientific | 01-812-25B | |
PVDF membrane | Fisher Scientific | IPVH00010 | |
Semken forceps | Fine Science Tools | 11008-13 | |
Small latex bulb, 2 mL | VWR | 82024-554 | |
Surgical scissors | Fine Science Tools | 14002-12 | |
Syringe filter, 25 mm, 0.2 μm pore size | Fisher Scientific | 09-740-108 | |
Syringe with luer tip, 10 mL | VWR | BD309604 | |
Transfer pipet | Fisher Scientific | 13-711-22 | |
Western blot cassette opening lever | Bio-Rad | 4560000 | |
Whatmann 3MM chr chromatography paper | Fisher Scientific | 05-714-5 | |
Reagents | |||
4-15% Precast protein gels, 10-well, 30 µL | Bio-Rad | 4561083 | |
β-glycerophosphate disodium salt hydrate | Sigma Aldrich | G5422-25G | stock concentration 1 M |
β-mercaptoethanol | Sigma Aldrich | M3148-100ML | |
Bovine serum albumin, fraction V, heat shock tested | Fisher Scientific | BP1600-100 | |
Bromophenol blue | Fisher Scientific | AC403140050 | |
Complete mini protease inhibitor cocktail | Sigma Aldrich | 11836153001 | stock concentration 25x |
DC protein assay kit II | Bio-Rad | 500-0112 | |
DMEM, high glucose | Gibco | 11965092 | |
E7, mouse monoclonal beta tubulin primary antibody, concentrate 0.1 mL | Developmental Studies Hybridoma Bank | E7 | 1:1,000 |
ECL western blotting substrate | Fisher Scientific | PI32106 | low picogram range |
ECL western blotting substrate | Genesee Scientific | 20-302B | low femtogram range |
Electrophoresis buffer, 5 L | Bio-Rad | 1610772 | stock concentration 10x |
Ethanol, 200 proof, 1 gallon | Decon Laboratories, Inc. | 2705HC | EtOH |
Ethylenediaminetetraacetic acid, Di Na salt dihydr. (crystalline powd./electrophor.) | Fisher Scientific | BP120-500 | EDTA |
Fetal bovine serum, characterized, US origin, 500 mL | HyClone | SH30071.03 | |
Glycerol (certified ACS) | Fisher Scientific | G33-4 | |
HRP-conjugated secondary antibody, goat anti-mouse IgG | Jackson ImmunoResearch Laboratories | 115-035-146 | 1:20,000 |
HRP-conjugated secondary antibody, goat anti-rabbit IgG | Jackson ImmunoResearch Laboratories | 111-035-003 | 1:20,000 |
Hydrochloric acid solution, 6N (certified) | Fisher Scientific | SA56-500 | HCl |
Igepal Ca – 630 non-ionic detergent | Fisher Scientific | ICN19859650 | Nonidet P-40 |
Isopropanol (HPLC) | Fisher Scientific | A451-1 | |
L-glutamine | Gibco | 25030081 | stock concentration 200 mM |
Methanol | Fisher Scientific | A454-4 | |
p44/42 MAPK (Erk1/2) primary antibody | Cell Signaling Technology | 9102S | 1:1,000; anti-Erk1/2 |
PDGF-BB recombinant ligand, rat | Fisher Scientific | 520BB050 | |
PDGF Receptor β primary antibody | Cell Signaling Technology | 3169S | 1:1,000 |
Penicillin-Streptomycin | Gibco | 15140122 | stock concentration 100 U/mL, 100 µg/mL |
Phenylmethanesulfonyl fluoride, 99% | Fisher Scientific | AC215740100 | PMSF; stock concentration 100 mM |
Phospho-p44/42 MAPK (Erk1/2) primary antibody | Cell Signaling Technology | 9101S | 1:1,000, anti-phospho-Erk1/2 |
Phospho-PDGF Receptor α /PDGF Receptor β primary antibody | Cell Signaling Technology | 3170S | 1:1,000 |
Potassium chloride (white crystals) | Fisher Scientific | BP366-500 | KCl |
Potassium phosphate monobasic (white crystals) | Fisher Scientific | BP362-500 | KH2PO4 |
SDS solution, 10% | Bio-Rad | 161-0416 | |
Sodium chloride (crystalline/biological,certified) | Fisher Scientific | S671-3 | NaCl |
Sodium fluoride (powder/certified ACS) | Fisher Scientific | S299-100 | NaF; aliquot for one time use; stock concentration 1 M |
Sodium orthovanadate, 99% | Fisher Scientific | AC205330500 | Na3VO4; stock concentration 100 mM |
Sodium phosphate dibasic anhydrous (granular or powder/certified ACS) | Fisher Scientific | S374-500 | Na2HPO4 |
Tissue culture PBS | Fisher Scientific | 21-031-CV | |
Transfer buffer, 5 L | Bio-Rad | 1610771 | stock concentration 10x |
Tris base (white crystals or crystalline powder/molecular biology) | Fisher Scientific | BP152-1 | |
Trypsin | BioWorld | 21560033 | |
Tween 20 | Fisher Scientific | BP337-500 | |
Western blot molecular weight marker | Bio-Rad | 1610374 | |
Software | |||
ImageJ software | National Institutes of Health (NIH) | ||
Animals | |||
Female 129S4 mice | gift of Dr. Philippe Soriano, Icahn School of Medicine at Mount Sinai |