Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

1. Co-immunoprecipitation

1. Seed HEK293 cells at 2 x 10⁵ cells per well in one 6-well plate in 2 ml Dulbecco's Modified Eagle's Medium (DMEM) supplement with 10% fetal bovine serum (FBS) and 100 U/mL Penicillin-Streptomycin (Pen-Strep).
2. Grow cells for 48 hr at 37 °C with 5% CO₂.
3. Wash cells with cold phosphate-buffered saline (PBS) three times. Harvest cells by centrifugation at 500 x g for 5 min at 4 °C.
4. Prepare nuclear and cytoplasmic fractions using commercial nuclear and cytoplasmic extraction reagents. Follow manufacturer's instruction with some modifications.
   1. Use 3 x 10⁶ cells for one co-immunoprecipitation experiment (approximately with 90% confluency from 3 wells of one 6-well plate). Add 300 µl cold cytoplasmic extraction solution (CER I) and vortex for 15 sec at the highest speed.
   2. Incubate for 30 min on ice. During incubation, vortex for 5 s every 10 min.
   3. Add 16.5 µl cold cytoplasmic extraction solution II (CER II), vortex for 5 sec and incubate for 5 min on ice.
   4. Vortex for 5 sec and centrifuge at 16,000 x g for 5 min at 4 °C.
   5. Transfer supernatant (cytoplasmic extract) into a new pre-chilled tube, and keep on ice until used.
   6. Wash insoluble pellets (containing nuclei) three times with 1 ml cold PBS each time by pipetting up and down five times. Remove PBS after wash.
   7. Add 150 µl cold nuclear extraction solution (NER), vortex for 15 sec and incubate 1 hr on ice. During incubation, vortex for 15 sec and pipette 10 times with a 200 µl tip every 10 min.
   8. Vortex for 15 s and centrifuge at 16,000 x g for 10 min at 4 °C.
   9. Transfer supernatant (nuclear extract) into a new pre-chilled tube, keep on ice until use.
5. Take out 10% of the volume of nuclear and cytoplasmic extracts each as inputs.
6. Add cold PBS to the remaining extracts to a final volume of 1 ml. Keep on ice until use.
   NOTE: Pre-clearing is not essential when using Protein G-conjugated magnetic beads. However, if the background is high, perform pre-clearing by incubating 40 µl Protein G-conjugated magnetic beads (50% slurry) and 1 ml diluted lysate from this step at 4 °C for 30 min on a rotator. Separate supernatant (pre-cleared lysate) from beads with magnetic rack. Discard the beads.
7. To couple primary antibody with Protein G-conjugated magnetic beads, incubate 40 µl Protein G-conjugated magnetic beads (50% slurry) with 4 µg rabbit polyclonal anti-RBM3 antibody or rabbit IgG (negative control) in 200 µl PBS plus Tween 20 buffer (PBST, 0.02% Tween 20) at room temperature (RT) for 40 min on a rotator.
8. Separate antibody-coupled beads and supernatant with a magnetic rack, and discard the supernatant. Wash the beads once with 200 µl PBST (0.02%).
9. Add 1 ml diluted lysates (or if necessary, pre-cleared lysates) to the beads, and incubate on a rotator at 4 °C overnight.
10. On the next day, separate the beads and supernatant by a magnetic rack, and discard the supernatant. Wash 3 x 10 min with 0.5 ml PBST (0.02%) for each tube at 4 °C on a rotator with a fixed speed of 20 rpm.
11. Elute proteins from the beads by adding 40 µl sample buffer (1x commercial sample buffer and 50 mM 1,4-dithiothreitol (DTT)). Heat at 70 °C for 10 min. Spin down and transfer liquids to a new 1.5 ml tube.
12. Dilute input lysates in sample buffer (final concentration: 1x commercial sample buffer and 50 mM DTT) and heat at 70 °C for 10 min. Load inputs and immunoprecipitated proteins from the last step to a precast 4-12% bis-Tris gel. The loading volume of each well should not exceed 20 µl. Perform electrophoresis in 1x commercial running buffer on ice for 35 min.
    NOTE: Load nuclear and cytoplasmic extracts in a ratio of 1:2 (V/V), which reflects the same initial amount of cells.
13. Transfer proteins to PVDF membrane in 1x commercial transfer buffer at 30 V for 2 hr at 4 °C.
14. Block membrane with 5% skimmed milk in PBST (0.1% Tween 20) at RT for 40 min on a shaker.
15. Incubate membrane with primary antibodies (both diluted in 1:1,000) in PBST (0.1%) at 4 °C overnight.
16. Wash 3 x 10 min with PBST (0.1%) at RT on a shaker.
17. Incubate membrane with horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit secondary antibodies (both diluted in 1:5,000) in PBST (0.1%) at RT for 1 hr.
18. Wash 3 x 10 min with PBST (0.1%) at RT on a shaker.
19. Use 0.2 ml enhanced chemiluminescence (ECL) substrate mixture per cm² membrane, incubate at RT for 5 min. Avoid all light from this step onwards except red lights.
20. Discard liquid and expose to an X-ray film in a film cassette. Develop film using an automatic film processing machine.

2. Immunocytochemistry and Proximity Ligation Assay

1. Seed HEK293 cells at 1.5 x 10⁴ cells per chamber in one 8-chamber poly-D-lysine coated slide in 0.4 ml DMEM supplemented with 10% FBS and 100 U/ml Pen-Strep.
2. Grow cells for 48 hr at 37 °C with 5% CO₂.
3. Aspirate medium, and fix cells with 4% paraformaldehyde (PFA) for 10 min at RT.
4. Aspirate PFA, and wash 3 x 10 min with 0.5 ml PBS per chamber.
5. Permeabilize and block cells with 0.5% Triton X-100 and 5% normal goat serum (NGS) in PBS at RT for 1 hr.
6. Incubate with primary antibodies in 0.1% Triton X-100 and 5% NGS in PBS on a shaker at 4 °C overnight. Dilute mouse monoclonal anti-NF90 antibody and rabbit polyclonal anti-RBM3 antibody 1:100 in PBS for double-staining. For negative control, omit independently either of the two primary antibodies, and omit both antibodies in a third control.
   NOTE: Do not use commercial blocking and diluting reagents.
7. Wash 3 x 10 min with 0.5 ml PBS per chamber.
8. Subject to immunocytochemistry or proximity ligation assay protocols
   1. Immunocytochemistry
      1. Incubate with 1:500 diluted green fluorescent dye-coupled anti-mouse and red fluorescent dye-coupled anti-rabbit secondary antibodies at RT for 1 hr. Avoid light from this step onwards.
      2. Counterstain nuclei with 4′, 6-diamidin-2-phenylindol (DAPI) diluted 1:5,000 in PBS at RT for 10 min.
      3. Wash 3 x 10 min with 0.5 ml PBS per chamber.
      4. Remove the chambers from the glass slide and dry. Mount with 250 µ, mounting medium per slide.
   2. Proximity ligation assay
      1. Prepare proximity ligation assay probes. Mix and dilute two proximity ligation assay probes (anti-mouse and anti-rabbit secondary antibodies attached with different oligonucleotides which can ligate through the addition of two other oligonucleotides in ligation solution) both 1:5 in 0.1% Triton X-100 and 5% NGS in PBS, with a total volume of 320 µl for one 8-chamber slide (approximately 40 µl per cm²). Incubate at RT for 20 min.
      2. Remove the chambers from the glass slide and add the diluted probes. Incubate in a humidity incubator at 37 °C for 1 hr.
      3. Prepare ligation solution. Mix 8 µl ligase, 64 µl 5 x ligation stock and 248 µl H₂O.
      4. Tap off the liquid from the slide, and wash 2 x 5 min in 1x Wash Buffer A (provided with the kit).
      5. Add ligation solution and incubate in a humidity incubator at 37 °C for 30 min.
      6. Prepare amplification solution. Mix 4 µl polymerase, 64 µl 5x amplification stock and 252 µl H₂O. Avoid light from this step onwards.
      7. Tap off liquid from the slide, and wash 2 x 2 min in 1x Wash Buffer A.
      8. Add the amplification solution and incubate in a dark humidity incubator at 37 °C for 100 min.
      9. Tap off the liquid from the slide, and wash 2 x 10 min in 1x Wash Buffer B (provided with the kit). Wash for 1 min in 0.01x Wash Buffer B.
      10. Dry the slide and mount with 250 µl of a commercial mounting medium (with DAPI) per slide.
9. Examine fluorescent signals under a microscope using a 10X eyepiece lens and 20X objective lens. Acquire images by a CCD camera.