Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex

Apoptosis initiation is mediated by activation of caspases at marker molecular platforms. We present the detection of the key initiation cell death complex DISC, which serves as a platform for caspase-8 activation. The advantage of this technique is that it allows the detection of the assembly and the composition of the DISC complex along with providing information on caspase-8 processing in this complex.

Begin the experiment with cells that are 80%to 90%confluent and adherent to the dish. Discard the medium and add fresh medium to the adherent cells. Then stimulate the cells with the selected concentration of CD95L by holding the plate at an angle and pipetting the ligand into the medium without touching the adherent cells.

To harvest the cells, place the cell dish on ice. Add 10 milliliters of cold PBS to the cell suspension and scrape the attached cells off the plate. Collect the cell suspension in a 50 milliliter tube.

Wash the cell dish with 10 milliliters of cold PBS twice and add the wash solution into the same 50 milliliter tube. Then centrifuge the cell suspension at 500 times G for five minutes at four degrees Celsius. After discarding the supernatant, resuspend the cell pellet in one milliliter of cold PBS.

Then transfer the cell suspension into a 1.5 milliliter tube. Centrifuge the cell suspension and resuspend the cell pellet in one milliliter of cold PBS again. Repeat the centrifugation once again, then resuspend the cell pellet in one milliliter of lysis buffer and incubate it for 30 minutes on ice.

After incubation, centrifuge the lysate at maximum speed for 15 minutes at four degrees Celsius, then transfer the supernatant to a clean tube. Discard the pellet and collect a 50 microliter sample of the lysate in another tube for determining the protein concentration. For immunoprecipitation, add two microliters of anti-APO1 antibody and 10 microliters of prepared Protein A Sepharose beads to the lysate.

Add 10 microliters of the beads alone to a separate tube containing the lysate to serve as a bead control. Incubate the mixture containing the lysate, the antibodies, and Protein A Sepharose beads overnight at four degrees Celsius with gentle mixing. The following day, centrifuge the mixture at 500 times G for four minutes at four degrees Celsius.

After discarding the supernatant, wash the beads by adding one milliliter of cold PBS and discarding the supernatant following centrifugation. Repeat this step at least three times. After discarding the last PBS wash, aspirate the beads preferably with a 50 microliter Hamilton syringe.

To perform the Western blot, add 20 microliters of 4X loading buffer to the beads and heat at 95 degrees Celsius for 10 minutes. Simultaneously, heat the lysate controls at 95 degrees Celsius for five minutes. Load the lysates, immunoprecipitants, and a protein standard onto a 12.5%SDS gel and run with a constant voltage of 80 volts.

After the gel run is complete, transfer the proteins from the SDS gel to a nitrocellulose membrane. After the transfer is complete, place the blotted membrane in a box and incubate it for an hour in blocking solution under gentle agitation, then wash the membrane three times for five minutes with PBST. To detect the proteins, add the first primary antibody at the indicated dilution to the membrane and incubate it overnight at four degrees Celsius with gentle agitation.

The next day, wash the membrane with three five-minute PBST washes, then incubate the membrane with 20 milliliters of secondary antibody with gentle shaking for one hour at room temperature. Wash the membrane three times with PBST again. After discarding the last PBST wash, add approximately one milliliter of horseradish peroxidase substrate to the membrane and detect the chemoluminescence signal.

Stimulation of cervical cancer HeLa-CD95 cells with CD95L resulted in a high level of CD95 DISC formation monitored via CD95 immunoprecipitation. CD95, FADD, procaspase-8, procaspase-10, and c-FLIPS were observed in these CD95 immunoprecipitations indicating efficient DISC formation. The cleavage products of p43-FLIP and p22-FLIP were detected in the immunoprecipitations, indicating caspase-8 activation.

HeLa-CD95 cells that over-express c-FLIP long were used for analysis of CD95 DISC formation. Similar to parental HeLa-CD95 cells, no recruitment of FADD, procaspase-9, procaspase-10, c-FLIP proteins, and PARP1 was detected in the immunoprecipitations from these cells without CD95L treatment. Activated primary T-cells were characterized by high levels of CD95, FADD, procaspase-8, procaspase-10, and c-FLIPS in anti-CD95 immunoprecipitations.

This technique allows the analysis of molecular DISC formation, including processing of caspase-8 at the DISC. All washing steps are very important. In addition, stimulation points and incubation times should be taken seriously.

This is the only way to measure the assembly on the DISC complex. However, to measure caspase-8 activation, one can also implement caspase-8 activity assays. This approach allowed us to get new insights into apoptosis initiation.