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

Summary

Here, an experimental workflow is presented that enables the detection of caspase-8 processing directly at the death-inducing signaling complex (DISC) and determines the composition of this complex. This methodology has broad applications, from unraveling the molecular mechanisms of cell death pathways to the dynamic modeling of apoptosis networks.

Abstract

Extrinsic apoptosis is mediated by the activation of death receptors (DRs) such as CD95/Fas/APO-1 or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-receptor 1/receptor 2 (TRAIL-R1/R2). Stimulation of these receptors with their cognate ligands leads to the assembly of the death-inducing signaling complex (DISC). DISC comprises DR, the adaptor protein Fas-associated protein with death domain (FADD), procaspases-8/-10, and cellular FADD-like interleukin (IL)-1β-converting enzyme-inhibitory proteins (c-FLIPs). The DISC serves as a platform for procaspase-8 processing and activation. The latter occurs via its dimerization/oligomerization in the death effector domain (DED) filaments assembled at the DISC.

Activation of procaspase-8 is followed by its processing, which occurs in several steps. In this work, an established experimental workflow is described that allows the measurement of DISC formation and the processing of procaspase-8 in this complex. The workflow is based on immunoprecipitation techniques supported by western blot analysis. This workflow allows careful monitoring of different steps of procaspase-8 recruitment to the DISC and its processing and is highly relevant for investigating molecular mechanisms of extrinsic apoptosis.

Introduction

One of the best-studied death receptors (DRs) is CD95 (Fas, APO-1). The extrinsic apoptotic pathway starts with the interaction of the DR with its cognate ligand, i.e., CD95L interacts with CD95 or TRAIL binds to TRAIL-Rs. This results in the formation of the DISC at the corresponding DR. DISC consists of CD95, FADD, procaspase-8/-10, and c-FLIP proteins^1,2. Furthermore, the DISC is assembled by interactions between death domain (DD)-containing proteins, such as CD95 and FADD, and DED-containing proteins such as FADD, procaspase-8/-10, and c-FLIP (Figure 1). Procaspase-8 undergoes oligomerization via association of its DEDs, resulting in the formation of DED filaments, followed by procaspase-8 activation and processing. This triggers a caspase cascade, which leads to cell death (Figure 1)^3,4. Thus, procaspase-8 is a central initiator caspase of the extrinsic apoptosis pathway mediated by CD95 or the TRAIL-Rs, activated at the corresponding macromolecular platform, DISC.

Two isoforms of procaspase-8, namely procaspase-8a (p55) and -8b (p53), are known to be recruited to the DISC⁵. Both isoforms comprise two DEDs. DED1 and DED2 are located at the N-terminal part of procaspase-8a/b followed by the catalytic p18 and p10 domains. Detailed cryo-electron microscopy (cryo-EM) analysis of procaspase-8 DEDs revealed the assembly of procaspase-8 proteins into filamentous structures called DED filaments^4,6. Remarkably, the linear procaspase-8 chains were initially suggested to be engaged in the dimerization followed by procaspase-8 activation at the DISC. Now, it is known that those chains are only a substructure of the procaspase-8 DED filament, the latter comprising three chains assembled into a triple helix^3,4,6,7.

Upon dimerization at the DED filament, conformational changes in procaspase-8a/b lead to the formation of the active center of procaspase-8 and its activation^3,8. This is followed by procaspase-8 processing, which is mediated via two pathways: the first one goes via the generation of a p43/p41 cleavage product and the second one via the initial generation of a p30 cleavage product. The p43/p41 pathway is initiated by the cleavage of procaspase-8a/b at Asp374, resulting in p43/p41 and p12 cleavage products (Figure 2). Further, these fragments are auto-catalytically cleaved at Asp384 and Asp210/216, giving rise to the formation of the active caspase-8 heterotetramer, p10₂/p18₂^9,10,11. In addition, it was shown that in parallel to the p43/p41 pathway of processing, procaspase-8a/b is also cleaved at Asp216, which leads to the formation of the C-terminal cleavage product p30, followed by its proteolysis to p10 and p18¹⁰ (Figure 2).

Procaspase-8a/b activation at the DED filament is strictly regulated by proteins named c-FLIPs¹². The c-FLIP proteins occur in three isoforms: c-FLIP_Long (c-FLIP_L), c-FLIP_Short (c-FLIP_S), and c-FLIP_Raji (c-FLIP_R). All three isoforms contain two DEDs in their N-terminal region. c-FLIP_L also has a C-terminal catalytically inactive caspase-like domain^12,13. Both short isoforms of c-FLIP-c-FLIP_S and c-FLIP_R-act in an anti-apoptotic manner by disrupting DED filament formation at the DISC^6,14,15. In addition, c-FLIP_L can regulate caspase-8 activation in a concentration-dependent manner. This can result in both pro- and anti-apoptotic effects^16,17,18. By forming the catalytically active procaspase-8/c-FLIP_L heterodimer, c-FLIP_L leads to the stabilization of the active center of procaspase-8 and its activation. The pro- or anti-apoptotic function of c-FLIP_L is directly dependent on its amount at the DED filaments and the subsequent amount of assembled procaspase-8/c-FLIP_L heterodimers¹⁹. Low or intermediate concentrations of c-FLIP_L at the DISC result in sufficient amounts of procaspase-8/c-FLIP_L heterodimers at the DED filament, which supports the activation of caspase-8. In contrast, increased amounts of c-FLIP_L directly lead to its anti-apoptotic effects at the DISC²⁰.

Taken together, the activation and processing of procaspase-8a/b at the DISC is a highly regulated process involving several steps. This paper discusses the measurement of procaspase-8 processing directly at the DISC as well as the analysis of the composition of this complex. This will be presented using CD95 DISC as the exemplary DR complex.

Protocol

​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 procedure²¹. 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 previously²⁰ or commercially available (see the Table of Materials)).
   ​NOTE: The concentration of the CD95L and the time of stimulation are cell-type dependent^{13,15,22,23,24,25}. 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 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 the 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 chemoluminescent signal (see the 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.

Results

To analyze caspase-8 recruitment to the DISC and its processing at the CD95 DISC, this paper describes a classical workflow, which combines IP of the CD95 DISC with western blot analysis. This allows the detection of several key features of caspase-8 activation at the DISC: the assembly of the caspase-8-activating macromolecular platform, recruitment of procaspase-8 to the DISC, and the processing of this initiator caspase (Figure 1 and Figure 2). This workflow involves the treatment of sensitive cells with CD95L in a time-dependent manner, followed by their lysis, immunoprecipitation using anti-CD95 (anti-APO-1) antibodies, and subsequent western blot analysis (Figure 3).

Cervical cancer HeLa-CD95 cells were used as an example to analyze the DISC formation²⁶. Stimulation of these cells with CD95L resulted in a high level of CD95 DISC formation, monitored via CD95-immunoprecipitation (Figure 4). CD95, FADD, procaspase-8, procaspase-10, and c-FLIPs were observed in these CD95-immunoprecipitations, indicating efficient DISC formation. Importantly, the cleavage products of procaspase-8a/b: p43/p41, p30, and p18 were detected at the DISC, which indicates activation of procaspase-8 and its subsequent processing. In particular, the cleavage products of procaspase-8 p43/p41 and p18 were detected, indicating the two aforementioned steps of the p43 processing pathway. In addition, the p30 product was detected, indicating the alternative pathway of caspase-8 processing. Furthermore, activation of procaspase-8 at the DISC is followed by the cleavage of its substrates such as c-FLIP proteins.

Indeed, the cleavage products of c-FLIP-p43-FLIP and p22-FLIP-were detected in the immunoprecipitations, indicating caspase-8 activation (Figure 4). Importantly, neither FADD, procaspase-8, procaspase-10, and c-FLIPs, nor their cleavage products were detected in the immunoprecipitation samples from untreated cells, which underlines the specificity of DISC-immunoprecipitation (Figure 4). Important information can be obtained from these experiments by quantifying the bands corresponding to the different cleavage products of procaspase-8 (Figure 4). This information can be used in the mathematical modeling of apoptosis networks and provides quantitative insights into pathway regulation.

The level of caspase-8 activation at the DISC is modulated by c-FLIPs. Hence, HeLa-CD95 cells that overexpress c-FLIP_L (HeLa-CD95-FL)¹⁵ were selected as the second example (Figure 5). The effects of the c-FLIP_L isoform in these experiments could be observed, resulting in a different rate of procaspase-8a/b processing to p43/p41 at the DISC compared to the corresponding proteolysis of procaspase-8 at the DISC in parental HeLa-CD95 cells, as described by Hillert et al.¹⁵. Similar to the observations in Figure 4, no recruitment of FADD, procaspase-8, procaspase-10, c-FLIP proteins, and their cleavage products was detected in the immunoprecipitations from HeLa-CD95-FL cells without CD95L treatment, which supports the specificity of these immunoprecipitations. More evidence for the specificity of immunoprecipitations is the absence of the recruitment of procaspase-3 and poly(ADP-ribose)polymerase 1 (PARP1) to the DISC, which was observed in the CD95L-treated immunoprecipitations (Figure 5). These proteins are not part of the complex, and their absence in the immunoprecipitation signals can serve as proof for the absence of nonspecific binding of abundant cellular proteins.

Finally, immunoprecipitations of CD95 DISC from suspension cells were performed as a third example, e.g., activated primary T cells (Figure 6). These cells are also characterized by high levels of CD95, FADD, procaspase-8, procaspase-10, and c-FLIPs that were observed in anti-CD95-immunoprecipitations along with their cleavage products (Figure 6). The detection of procaspase-8 cleavage products in the immunoprecipitations indicates the activation and processing of this initiator caspase in the DISC-immunoprecipitation from primary T cells. These experiments indicate that the DISC can be immunoprecipitated from many adherent and suspension cells and that caspase-8 processing and activation can be validated by western blot analysis.

Figure 1: Schematic presentation of the CD95 signaling pathway. CD95L triggers the DISC assembly. The DISC comprises CD95, FADD, procaspase-8/-10, and c-FLIP. FADD binds to CD95 via its DD, whereas procaspase-8, procaspase-10, and c-FLIPs interact via their DEDs, forming DED filaments. Formation of the DED filaments serves as a platform for procaspase-8 dimerization, processing, and subsequent activation. The active caspase-8 heterotetramer, p18₂/p10₂, activates caspase-3 by cleavage, which leads to apoptosis. Abbreviations: CD = cluster of differentiation; CD95L = CD95 ligand; DISC = death-inducing signaling complex; DD = death domain; FADD= Fas-associated death domain; c-FLIP = cellular FADD-like interleukin (IL)-1β-converting enzyme-inhibitory protein; DED = death effector domain; c-FLIP_L = c-FLIP_Long. Please click here to view a larger version of this figure.

Figure 2: Procaspase-8 processing at the DISC. Two ways of procaspase-8a/b processing at the DISC are shown. The first way involves p43/p41 generation followed by p18 formation. The second way involves p30 generation followed by its processing to p18 and p10. The residues are numbered according to the sequence of procaspase-8a. Abbreviations: DED = death effector domain. Please click here to view a larger version of this figure.

Figure 3: Schematic presentation of the experimental setup of the DISC-IP. Cells are stimulated with CD95L. After stimulation, the cells are harvested and collected, followed by different washing steps. The cells are then lysed, and the lysates are collected. Subsequently, protein A-sepharose beads and anti-APO-1 (anti-CD95) antibodies were added to the lysate and incubated overnight. After several washing steps, the immunoprecipitations were analyzed by western blotting. Abbreviations: DISC = death-inducing signaling complex; IP = immunoprecipitation; CD95L = CD95 ligand. Please click here to view a larger version of this figure.

Figure 4: CD95 DISC formation in HeLa-CD95 cells. HeLa-CD95 cells were stimulated with 125 ng/mL CD95L for 30 min or 1 h. CD95 DISC-IPs were carried out using anti-APO-1 (anti-CD95) antibodies. The composition of the IPs was examined by western blot analysis using the antibodies for the indicated proteins. Actin was used as a loading control. Inputs are shown. Quantification of procaspase-8 cleavage products at the DISC is shown and normalized to CD95 signal. Abbreviations: l.e. = long exposure; s.e. = short exposure; BC = control IP with 'beads-only', without the addition of antibodies; CD95L = CD95 ligand; DISC = death-inducing signaling complex; IP = immunoprecipitation; FADD = Fas-associated death domain; c-FLIP = cellular FADD-like interleukin (IL)-1β-converting enzyme-inhibitory protein; c-FLIP_L = c-FLIP_Long; c-FLIP_S = c-FLIP_Short; M = molecular weight in kiloDalton (kDa). Please click here to view a larger version of this figure.

Figure 5: CD95 DISC formation in c-FLIP_L-overexpressing HeLa-CD95 cells. HeLa-CD95-FL cells were stimulated with 250 ng/mL CD95L for the indicated time points (1-3 h). CD95 DISC-IPs were carried out using anti-APO-1 (anti-CD95) antibodies. The composition of the IPs was examined by western blot analysis and analyzed for the indicated proteins. Actin was used as a loading control. Inputs are shown. Quantification of procaspase-8 cleavage products at the DISC is shown and normalized to the CD95 signal. One representative experiment out of two is shown. Abbreviations: l.e. = long exposure; s.e. = short exposure; BC = control IP with 'beads-only', without the addition of antibodies; CD95L = CD95 ligand; DISC = death-inducing signaling complex; IP = immunoprecipitation; FADD = Fas-associated death domain; c-FLIP = cellular FADD-like interleukin (IL)-1β-converting enzyme-inhibitory protein; c-FLIP_L = c-FLIP_Long; PARP1 = poly(ADP-ribose)polymerase 1; M = molecular weight in kiloDalton (kDa). Please click here to view a larger version of this figure.

Figure 6: CD95 DISC formation in primary T cells. Primary activated T cells were stimulated with 500 ng/mL CD95L for 15 min and 30 min. CD95 DISC-IPs were carried out using anti-APO-1 (anti-CD95) antibodies. The composition of the IPs was examined by western blot analysis and analyzed for the indicated proteins. Actin was used as a loading control. Quantification of procaspase-8 cleavage products at the DISC is shown and normalized to the CD95 signal. Inputs are shown. Abbreviations: l.e. = long exposure; s.e. = short exposure; ; CD95L = CD95 ligand; DISC = death-inducing signaling complex; IP = immunoprecipitation; FADD = Fas-associated death domain; c-FLIP = cellular FADD-like interleukin (IL)-1β-converting enzyme-inhibitory protein; c-FLIP_L = c-FLIP_Long; M = molecular weight in kiloDalton (kDa). Please click here to view a larger version of this figure.

Discussion

This approach was first described by Kischkel et al.²⁷ and has successfully been developed since then by several groups. Several important issues have to be considered for efficient DISC-immunoprecipitation and monitoring caspase-8 processing in this complex.

First, it is essential to follow all washing steps during immunoprecipitation. Especially important are the final washing steps of the sepharose beads and the drying of the sepharose beads. This must be done correctly to increase the signal/noise ratio of immunoprecipitation, allowing the detection of caspase-8 recruitment and processing at the DISC. Importantly, for very sensitive analytical techniques, such as mass spectrometry, a "preclearing step," which includes the incubation of the lysates with only the sepharose beads or isotype control antibodies, can also be important in reducing the noise. However, several studies have shown that this preclearing step is not essential for the detection of caspase-8 recruitment to the DISC by western blotting^7,23. However, as mentioned, the washing of the beads at the end of immunoprecipitation is essential for obtaining reliable results. Nonspecific binding of abundant cellular proteins to the sepharose beads can essentially decrease the specific signals of the core DISC components. As an important control for the absence of the nonspecific binding, western blot analysis of the proteins, reported not to be present at the DISC, might be performed. An example of this analysis is given in Figure 5, in which the recruitment of PARP1 and caspase-3 to the DISC-immunoprecipitation was not observed. This indicates the specificity of the particular immunoprecipitation and sufficient washing of the sepharose beads during the experiment.

Second, it is crucial to perform negative controls such as a beads-only control or an immunoprecipitation control with an antibody with the same isotype as the antibody used for the immunoprecipitation. For anti-APO-1 antibodies, anti-mouse IgG3 antibodies can be used as an isotype control. Third, it is important to monitor the results of the immunoprecipitation from untreated samples, in which only CD95 should be observed. The detection of FADD, c-FLIP, or procaspase-8 in these samples typically indicates the presence of some shortcomings in the immunoprecipitation protocol or washing steps. This could give rise to assumptions on the stimulation-independent association of FADD or c-FLIP with CD95, which might be not entirely correct, and instead indicate flaws in immunoprecipitation. Fourth, for each immunoprecipitation, the inputs or lysates must be carefully analyzed in parallel to measure the expression and posttranslational modifications of the core proteins analyzed by immunoprecipitation.

Fifth, time-dependent analysis allows changes in the complex to be followed over time and provides yet another important confirmation on the specificity of the proteins recruited to the complex. In this regard, an important issue is that each cell line has a different level of CD95 expression and of intracellular components of this complex. Accordingly, the exact timing of the CD95 DISC formation has to be carefully established for each particular cell type. Finally, the crucial issue for the analysis of the DISC dynamics is the comparison of the amount of protein in each immunoprecipitation. For CD95 DISC-immunoprecipitations performed using anti-APO-1 antibodies, the amount of CD95 is a key measure of the equal amount of the complexes being compared. This might be difficult because the intensity of the CD95 signal in the immunoprecipitations is relatively high. However, one must find the optimal time interval for measuring the corresponding western blot signal. Another obstacle is that CD95 is a highly glycosylated protein, which also contributes to the difficulties in its detection in immunoprecipitation due to the presence of a particular pattern of several 'blurry' bands²⁸.

DISC-immunoprecipitation analysis provides an optimal basis for detecting caspase activation and processing. Indeed, immunoprecipitation combined with western blotting allows for quantitative detection of cleavage products of procaspase-8a/b: p43/p41, p30, and p18, as shown in this study (Figure 4, Figure 5, and Figure 6). This, in turn, enables experimenters to follow changes in procaspase-8a/b processing over time and distinguish different cleavage steps of procaspase-8. This approach has been successfully used to describe caspase-8 activation in mathematical models and distinguish between inter- and intramolecular procaspase-8 processing at the DISC^7,20,29. Moreover, measuring caspase-8 cleavage products by western blotting has clear advantages compared to the conventional caspase-8 activity assays based on IETD substrate. In the latter case, it is well established that IETD also serves as a substrate for the other caspases. Hence, there is increasing evidence that the detection of IETD activity indicates a general increase of caspase activity in the cell. In contrast, using western blot analysis can help specifically assign the corresponding bands in the western blot to caspase-8, allowing the researcher to be confident that caspase-8 is activated in this complex. Furthermore, as mentioned above, measuring caspase-8 processing at the DISC presents an excellent tool for mathematical modeling and systems biology studies. Taken together, a classical workflow is presented to allow the monitoring of different steps of procaspase-8 activation and processing, which is essential for unraveling the molecular mechanisms of cell death.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
12.5% SDS gel	self made		for two separating gels: 3.28 mL distilled H2O 2.5 mL Tris; pH 8.8; 1.5 M 4.06 mL acrylamide 100 µL 10% SDS 100 µL 10% APS 7.5 µL TEMED for two collecting gels: 3.1 mL distilled H2O 1.25 mL Tris; pH 6.8; 1.5 M 0.5 mL acrylamide 50 µL 10% SDS 25 µL 10% APS 7.5 µL TEMED
14.5 cm cell dishes	Greiner	639160
acrylamide	Carl Roth	A124.1
Aerosol filter pipet tips with high recovery filter and wide orifice	VWR	46620-664 (North America) or 732-0559 (Europe)	Used for pipetting beads to the lysate
anti-actin Ab	Sigma Aldrich	A2103	dilution: 1:4000 in PBST + 1:100 NaN3
anti-APO-1 Ab	provided in these experiments by Prof. P. Krammer or can be purchased by Enzo	ALX-805-038-C100	used only for immunoprecipitation
anti-caspase-10 Ab	Biozol	MBL-M059-3	dilution: 1:1000 in PBST + 1:100 NaN3
anti-caspase-3 Ab	cell signaling	9662 S	dilution: 1:2000 in PBST + 1:100 NaN3
anti-caspase-8 Ab C15	provided in these experiments by Prof. P. Krammer or can be purchased by ENZO	ALX-804-242-C100	dilution: 1:20 in PBST + 1:100 NaN3
anti-CD95 Ab	Santa Cruz	sc-715	dilution: 1:2000 in PBST + 1:100 NaN3
anti-c-FLIP NF6 Ab	provided in these experiments by Prof. P. Krammer or can be purchased by ENZO	ALX-804-961-0100	dilution: 1:10 in PBST + 1:100 NaN3
anti-FADD 1C4 Ab	provided in these experiments by Prof. P. Krammer or can be purchased by ENZO	ADI-AAM-212-E	dilution: 1:10 in PBST + 1:100 NaN3
anti-PARP Ab	cell signaling	9542	dilution: 1:1000 in PBST + 1:100 NaN3
APS	Carl Roth	9592.3
β-mercaptoethanol	Carl Roth	4227.2
Bradford solution Protein Assay Dye Reagent Concentrate 450ml	Bio Rad	500-0006	used according to manufacturer's instructions
CD95L	provided in these experiments by Prof. P. Krammer or can be purchased by ENZO	ALX-522-020-C005
chemoluminescence detector Chem Doc XRS+	Bio Rad
cOmplete Protese Inhibitor Cocktail (PIC)	Sigma Aldrich	11 836 145 001	prepared according to manufacturer's instructions
DPBS (10x) w/o Ca, Mg	PAN Biotech	P04-53500	dilution 1:10 with H2O, storage in the fridge
eletrophoresis buffer	self made		10x electrophoresis buffer: 60.6 g Tris 288 g glycine 20 g SDS ad 2 L H2O 1:10 dilution before usage
glycine	Carl Roth	3908.3
Goat Anti-Mouse IgG1 HRP	SouthernBiotech	1070-05	dilution 1:10.000 in PBST + 5% milk
Goat Anti-Mouse IgG2b HRP	SouthernBiotech	1090-05	dilution 1:10.000 in PBST + 5% milk
Goat Anti-Rabbit IgG-HRP	SouthernBiotech	4030-05	dilution 1:10.000 in PBST + 5% milk
Interleukin-2 Human(hIL-2)	Merckgroup/ Roche	11011456001	for activation of T cells
KCl	Carl Roth	6781.2
KH2PO4	Carl Roth	3904.1
loading buffer 4x Laemmli Sample Buffer,10 mL	Bio Rad	161-0747	prepared according to manufacturer's instructions
Luminata Forte Western HRP substrate	Millipore	WBLUFO500
lysis buffer	self made		13.3 mL Tris-HCl; pH 7.4; 1.5 M 27.5 mL NaCl; 5 M 10 mL EDTA; 2 mM 100 mL Triton X-100 add 960 mL H2O
medium for adhaerent cells DMEM F12 (1:1) w stable Glutamine,  2,438 g/L	PAN Biotech	P04-41154	adding 10% FCS, 1% Penicillin-Streptomycin and 0.0001% Puromycin to the medium
medium for primary T cells	gibco by Life Technologie	21875034	adding 10% FCS and 1% Penicillin-Streptomycin to the medium
milk powder	Carl Roth	T145.4
Na2HPO4	Carl Roth	P030.3
NaCl	Carl Roth	3957.2
PBST	self made		20x PBST: 230 g NaCl 8 g KCl 56.8 g Na2HPO4 8 g KH2PO4 20 mL Tween-20 ad 2 L H2O dilution 1:20 before usage
PBST + 5% milk	self made		50 g milk powder + 1 L PBST
PHA	Thermo Fisher Scientific	R30852801	for activation of T Cells
Power Pac HC	Bio Rad
Precision Plus Protein Standard All Blue	Bio Rad	161-0373	use between 3-5 µL
Protein A Sepharose CL-4B beads	Novodirect/ Th.Geyer	GE 17-0780-01	affinity resin beads prepared according to manufacturer's instructions
scraper	VWR	734-2602
SDS	Carl Roth	4360.2
shaker	Heidolph
sodium azide	Carl Roth	K305.1
TEMED	Carl Roth	2367.3
Trans Blot Turbo mini-size transfer stacks	Bio Rad	170-4270	used according to manufacturer's instructions
TransBlot Turbo 5x Transfer Buffer	Bio Rad	10026938	prepared according to manufacturer's instructions
TransBlot Turbo Mini-size nictrocellulose membrane	Bio Rad	170-4270	used according to manufacturer's instructions
Trans-Blot-Turbo	Bio Rad
Tris	Chem Solute	8,08,51,000
Triton X-100	Carl Roth	3051.4
Tween-20	Pan Reac Appli Chem	A4974,1000