Chemiluminescent Western Blot Assay for Protein Processing

T-cell experiments were performed according to the ethical agreement 42502-2-1273 Uni MD.

1. Preparing cells for the experiment

NOTE: The average number of cells for this immunoprecipitation is 1 × 10⁷. Adherent cells have to be seeded one day before the experiment so that there are 1 × 10⁷ cells on the day of the experiment.

1. Preparing adherent cells for the experiment
   1. Seed 5-8 × 10⁶ adherent cells in 20 mL of medium (see the Table of Materials for the composition) for each condition in 14.5 cm dishes one day before the experiment starts.
   2. On the day of the experiment, ensure that the cells are 80-90% confluent and adherent to the dish. Discard the medium and add fresh medium to the adherent cells.
2. Preparing suspension cells for the experiment
   1. Carefully place 1 × 10⁷ suspension cells in 10 mL of culture medium (see the Table of Materials for the composition) per condition in 14.5 cm dishes immediately before the experiment starts.
   2. If using primary cells, isolate primary T cells according to the previously described procedure. Treat primary T cells with 1 µg/mL phytohemagglutinin for 24 h, followed by 25 U/mL IL2 treatment for 6 days.
   3. Carefully place 1 × 10⁸ primary T cells in 10 mL of culture medium (see the Table of Materials for the composition) per condition in 14.5 cm dishes immediately before the experiment starts.
      NOTE: This higher number of primary T cells is recommended, as these cells are smaller.

2. CD95L stimulation

1. Stimulate the cells with CD95L (produced as described previously or commercially available (see the Table of Materials).
   NOTE: The concentration of the CD95L and the time of stimulation are cell-type dependent. Prepare one stimulation condition twice to generate a 'bead control' sample in parallel.
   1. Stimulate adherent cells with the selected concentration of CD95L. Hold the plate at an angle and pipet the ligand into the medium without touching the adherent cells.
   2. Stimulate suspension cells with CD95L by pipetting the ligand solution into the cell suspension.

3. Cell harvest and lysis

1. Place the cell dishes on ice.
   NOTE: Do not discard the medium. Dying cells float in the medium and are important for the analysis.
2. Add 10 mL of cold phosphate-buffered saline (PBS) to the cell suspension and scrape the attached cells off the plate. Collect the cell suspension in a 50 mL tube.
3. Wash the cell dish with 10 mL of cold PBS twice and place the wash solution into the same 50 mL tube. Centrifuge the cell suspension at 500 × g for 5 min, 4 °C.
4. Discard the supernatant and resuspend the cell pellet with 1 mL of cold PBS. Transfer the cell suspension into a 1.5 mL tube.
5. Centrifuge the cell suspension at 500 × g for 5 min, 4 °C. Discard the supernatant and resuspend the cell pellet with 1 mL of cold PBS.
6. Centrifuge the cell suspension at 500 × g for 5 min, 4 °C. Discard the supernatant and resuspend the cell pellet with 1 mL of lysis buffer (containing 4% protease inhibitor cocktail). Incubate it for 30 min on ice.
7. Centrifuge the lysate at maximal speed (~17,000 × g) for 15 min, 4 °C.
8. Transfer the supernatant (lysate) to a clean tube. Discard the pellet. Take 50 µL of the lysate in another tube. Analyze the protein concentration by Bradford assay and take the amount of lysate corresponding to 25 µg of protein in a vial. Add loading buffer (see the Table of Materials for the composition) to the vial. Store it at -20 °C as lysate control.

4. Immunoprecipitation (IP)

1. Add 2 µL of anti-APO-1 antibodies and 10 µL of protein A sepharose beads (prepared as recommended by the manufacturer) to the lysate. Add only 10 µL of the beads to a separate tube containing lysate (stimulated sample) to generate a 'bead control.'
   NOTE: Use pipet tips with wide orifices either by cutting the tips or buying special tips for IP while handling the protein A sepharose beads.
2. Incubate the mixture of lysate with antibodies/protein A sepharose beads with gentle mixing overnight at 4 °C. Centrifuge the lysates with antibodies/protein A sepharose beads at 500 × g for 4 min, 4 °C. Discard the supernatant, add 1 mL of cold PBS to the beads, and repeat this step at least three times.
3. Discard the supernatant. Aspirate the beads preferably with a 50 µL Hamilton syringe.

5. Western blot

1. Add 20 µL of 4x loading buffer (see the Table of Materials for the composition) to the beads and heat at 95 °C for 10 min. Heat the lysate controls at 95 °C for 5 min.
2. Load the lysates, IPs, and a protein standard onto a 12.5% sodium dodecyl sulfate (SDS) gel (see the Table of Materials for the gel preparation) and run with a constant voltage of 80 V.
3. Transfer the proteins from the SDS gel to a nitrocellulose membrane.
   NOTE: Here, the semi-dry technique, optimized for the proteins of interest, was used for the transfer over 12 min (25 V; 2.5 A= constant). Soak the nitrocellulose membrane and two mini-size transfer stacks in electrophoresis buffer (see the Table of Materials for the composition, prepare according to the manufacturer's instructions) for a few minutes before western blotting.
4. Place the blotted membrane in a box and block it for 1 h in a blocking solution (0.1% Tween-20 in PBS (PBST) + 5% milk). Incubate the membrane with the blocking solution under gentle agitation.
5. Wash the membrane three times with PBST for 5 min each wash.

6. Western blot detection

1. Add the first primary antibody at the indicated dilution (see Table of Materials) to the membrane and incubate it overnight at 4 °C with gentle agitation.
2. Wash the membrane three times with PBST for 5 min each wash.
3. Incubate the membrane with 20 mL of secondary antibody (diluted 1:10,000 in PBST + 5% milk) with gentle shaking for 1 h at room temperature.
4. Wash the membrane three times with PBST for 5 min each wash.
5. Discard PBST and add approximately 1 mL of horseradish peroxidase substrate to the membrane.
6. Detect the chemiluminescent signal (see Table of Materials).
   NOTE: The exposure time and the number of captured images depend on the amount of protein in the cell and the specificity of the antibodies used. It must be established empirically for each antibody used for the detection.