Name: measuring composition of cd95 death-inducing signaling complex and processing of procaspase-8 in this complex
Uploaded: 2021-08-02T15:00:00
Description: Watch this Scientific Journal Video about Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex at JoVE.com

We acknowledge the Wilhelm Sander-Foundation (2017.008.02), the Center of Dynamic Systems (CDS), funded by the EU-program ERDF (European Regional Development Fund) and the DFG (LA 2386) for supporting our work. We thank Karina Guttek for supporting our experiments. We acknowledge Prof. Dirk Reinhold (OvGU, Magdeburg) for providing us primary T cells.

&#8203;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 &#215; 107. Adherent cells have to be seeded one day before the experiment so that there are 1 &#215; 107 cells&#160;on the day of the experiment.
<ol>
	<li>Preparing adherent cells for the experiment
	<ol>
		<li>Seed 5-8 &#215; 10<...

<ol>
	<li>Lavrik, I. N., Krammer, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+CD95/Fas+signaling+at+the+DISC.">Regulation of CD95/Fas signaling at the DISC.</a> Cell Death and Differentiation. 19 (1), 36-41 (2012).</li><li>Krammer, P. H., Arnold, R., Lavrik, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+and+death+in+peripheral+T+cells.">Life and death in peripheral T cells.</a> Nature Reviews Immunology. 7 (7), 532-542 (2007).</li><li>Dickens, L. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+death+effector+domain+chain+DISC+model+reveals+a+crucial+role+for+caspase-8+chain+assembly+in+mediating+apoptotic+cell+death.">A death effector domain chain DISC model reveals a crucial role for caspase-8 chain assembly in mediating apoptotic cell death.</a> Molecular Cell. 47 (2), 291-305 (2012).</li><li>Fu, T. -. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryo-EM+structure+of+caspase-8+tandem+DED+filament+reveals+assembly+and+regulation+mechanisms+of+the+death-inducing+signaling+complex.">Cryo-EM structure of caspase-8 tandem DED filament reveals assembly and regulation mechanisms of the death-inducing signaling complex.</a> Molecular Cell. 64 (2), 236-250 (2016).</li><li>Scaffidi, C., Medema, J. P., Krammer, P. H., Peter, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FLICE+is+predominantly+expressed+as+two+functionally+active+isoforms,+caspase-8/a+and+caspase-8/b.">FLICE is predominantly expressed as two functionally active isoforms, caspase-8/a and caspase-8/b.</a> Journal of Biological Chemistry. 272 (43), 26953-26958 (1997).</li><li>Fox, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryo-EM+structural+analysis+of+FADD:Caspase-8+complexes+defines+the+catalytic+dimer+architecture+for+co-ordinated+control+of+cell+fate.">Cryo-EM structural analysis of FADD:Caspase-8 complexes defines the catalytic dimer architecture for co-ordinated control of cell fate.</a> Nature Communications. 12 (1), 1-17 (2021).</li><li>Schleich, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stoichiometry+of+the+CD95+death-inducing+signaling+complex:+experimental+and+modeling+evidence+for+a+death+effector+domain+chain+model.">Stoichiometry of the CD95 death-inducing signaling complex: experimental and modeling evidence for a death effector domain chain model.</a> Molecular Cell. 47 (2), 306-319 (2012).</li><li>Hughes, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+the+death-inducing+signaling+complex+reveals+a+substrate+switch+that+determines+CD95-mediated+death+or+survival.">Reconstitution of the death-inducing signaling complex reveals a substrate switch that determines CD95-mediated death or survival.</a> Molecular Cell. 35 (3), 265-279 (2009).</li><li>Lavrik, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+active+caspase-8+heterotetramer+is+formed+at+the+CD95+DISC+[2].">The active caspase-8 heterotetramer is formed at the CD95 DISC [2].</a> Cell Death and Differentiation. 10 (1), 144-145 (2003).</li><li>Hoffmann, J. C., Pappa, A., Krammer, P. H., Lavrik, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+C-terminal+cleavage+product+of+procaspase-8,+p30,+defines+an+alternative+pathway+of+procaspase-8+activation.">A new C-terminal cleavage product of procaspase-8, p30, defines an alternative pathway of procaspase-8 activation.</a> Molecular and Cellular Biology. 29 (16), 4431-4440 (2009).</li><li>Golks, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+CAP3+in+CD95+signaling:+New+insights+into+the+mechanism+of+procaspase-8+activation.">The role of CAP3 in CD95 signaling: New insights into the mechanism of procaspase-8 activation.</a> Cell Death and Differentiation. 13 (3), 489-498 (2006).</li><li>Ozt&#252;rk, S., Schleich, K., Lavrik, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+FLICE-like+inhibitory+proteins+(c-FLIPs):+Fine-tuners+of+life+and+death+decisions.">Cellular FLICE-like inhibitory proteins (c-FLIPs): Fine-tuners of life and death decisions.</a> Experimental Cell Research. 318 (11), 1324-1331 (2012).</li><li>Golks, A., Brenner, D., Fritsch, C., Krammer, P. H., Lavrik, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=c-FLIPR,+a+new+regulator+of+death+receptor-induced+apoptosis.">c-FLIPR, a new regulator of death receptor-induced apoptosis.</a> Journal of Biological Chemistry. 280 (15), 14507-14513 (2005).</li><li>Hughes, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-operative+and+hierarchical+binding+of+c-FLIP+and+caspase-8:+A+unified+model+defines+how+c-FLIP+isoforms+differentially+control+cell+fate.">Co-operative and hierarchical binding of c-FLIP and caspase-8: A unified model defines how c-FLIP isoforms differentially control cell fate.</a> Molecular Cell. 61 (6), 834-849 (2016).</li><li>Hillert, L. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+and+short+isoforms+of+c-FLIP+act+as+control+checkpoints+of+DED+filament+assembly.">Long and short isoforms of c-FLIP act as control checkpoints of DED filament assembly.</a> Oncogene. 39 (8), 1756-1772 (2020).</li><li>Yu, J. W., Jeffrey, P. D., Shi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+procaspase-8+activation+by+c-FLIPL.">Mechanism of procaspase-8 activation by c-FLIPL.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (20), 8169-8174 (2009).</li><li>Micheau, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+long+form+of+FLIP+is+an+activator+of+caspase-8+at+the+Fas+death-inducing+signaling+complex.">The long form of FLIP is an activator of caspase-8 at the Fas death-inducing signaling complex.</a> Journal of Biological Chemistry. 277 (47), 45162-45171 (2002).</li><li>Chang, D. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C-FLIPL+is+a+dual+function+regulator+for+caspase-8+activation+and+CD95-mediated+apoptosis.">C-FLIPL is a dual function regulator for caspase-8 activation and CD95-mediated apoptosis.</a> EMBO Journal. 21 (14), 3704-3714 (2002).</li><li>Hillert, L. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+DISC+regulation+via+pharmacological+targeting+of+caspase-8/c-FLIPL+heterodimer.">Dissecting DISC regulation via pharmacological targeting of caspase-8/c-FLIPL heterodimer.</a> Cell Death and Differentiation. 27 (7), 2117-2130 (2020).</li><li>Fricker, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model-based+dissection+of+CD95+signaling+dynamics+reveals+both+a+pro-+and+antiapoptotic+role+of+c-FLIPL.">Model-based dissection of CD95 signaling dynamics reveals both a pro- and antiapoptotic role of c-FLIPL.</a> Journal of Cell Biology. 190 (3), 377-389 (2010).</li><li>Arndt, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+TCR+activation+kinetics+in+primary+human+T+cells+upon+focal+or+soluble+stimulation.">Analysis of TCR activation kinetics in primary human T cells upon focal or soluble stimulation.</a> Journal of Immunological Methods. 387 (1-2), 276-283 (2013).</li><li>Pietkiewicz, S., Eils, R., Krammer, P. H., Giese, N., Lavrik, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+treatment+of+CD95L+and+gemcitabine+in+pancreatic+cancer+cells+induces+apoptotic+and+RIP1-mediated+necroptotic+cell+death+network.">Combinatorial treatment of CD95L and gemcitabine in pancreatic cancer cells induces apoptotic and RIP1-mediated necroptotic cell death network.</a> Experimental Cell Research. 339 (1), 1-9 (2015).</li><li>Schleich, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+architecture+of+the+DED+chains+at+the+DISC:+regulation+of+procaspase-8+activation+by+short+DED+proteins+c-FLIP+and+procaspase-8+prodomain.">Molecular architecture of the DED chains at the DISC: regulation of procaspase-8 activation by short DED proteins c-FLIP and procaspase-8 prodomain.</a> Cell Death and Differentiation. 23 (4), 681-694 (2016).</li><li>Lavrik, I. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+CD95+threshold+signaling:+Triggering+of+CD95+(FAS/APO-1)+at+low+concentrations+primarily+results+in+survival+signaling.">Analysis of CD95 threshold signaling: Triggering of CD95 (FAS/APO-1) at low concentrations primarily results in survival signaling.</a> Journal of Biological Chemistry. 282 (18), 13664-13671 (2007).</li><li>Sprick, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase-10+is+recruited+to+and+activated+at+the+native+TRAIL+and+CD95+death-inducing+signalling+complexes+in+a+FADD-dependent+manner+but+can+not+functionally+substitute+caspase-8.">Caspase-10 is recruited to and activated at the native TRAIL and CD95 death-inducing signalling complexes in a FADD-dependent manner but can not functionally substitute caspase-8.</a> EMBO Journal. 21 (17), 4520-4530 (2002).</li><li>Neumann, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+within+the+CD95+death-inducing+signaling+complex+decide+life+and+death+of+cells.">Dynamics within the CD95 death-inducing signaling complex decide life and death of cells.</a> Molecular Systems Biology. 6 (1), 352 (2010).</li><li>Kischkel, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxicity-dependent+APO-1+(Fas/CD95)-associated+proteins+form+a+death-inducing+signaling+complex+(DISC)+with+the+receptor.">Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor.</a> EMBO Journal. 14 (22), 5579-5588 (1995).</li><li>Seyrek, K., Richter, M., Lavrik, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decoding+the+sweet+regulation+of+apoptosis:+the+role+of+glycosylation+and+galectins+in+apoptotic+signaling+pathways.">Decoding the sweet regulation of apoptosis: the role of glycosylation and galectins in apoptotic signaling pathways.</a> Cell Death and Differentiation. 26 (6), 981-993 (2019).</li><li>Kallenberger, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-+and+interdimeric+caspase-8+self-cleavage+controls+strength+and+timing+of+CD95-induced+apoptosis.">Intra- and interdimeric caspase-8 self-cleavage controls strength and timing of CD95-induced apoptosis.</a> Science Signaling. 7 (316), 23 (2014).</li></ol>

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 correc...

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 proteins1,2. 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 filaments4,6. 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 helix3,4,6,7.
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 activation3,8. 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/p1829,10,11. 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-FLIPLong (c-FLIPL), c-FLIPShort (c-FLIPS), and c-FLIPRaji (c-FLIPR). All three isoforms contain two DEDs in their N-terminal region. c-FLIPL also has a C-terminal catalytically inactive caspase-like domain12,13. Both short isoforms of c-FLIP-c-FLIPS and c-FLIPR-act in an anti-apoptotic manner by disrupting DED filament formation at the DISC6,14,15. In addition, c-FLIPL can regulate caspase-8 activation in a concentration-dependent manner. This can result in both pro- and anti-apoptotic effects16,17,18. By forming the catalytically active procaspase-8/c-FLIPL heterodimer, c-FLIPL leads to the stabilization of the active center of procaspase-8 and its activation. The pro- or anti-apoptotic function of c-FLIPL is directly dependent on its amount at the DED filaments and the subsequent amount of assembled procaspase-8/c-FLIPL heterodimers19. Low or intermediate concentrations of c-FLIPL at the DISC result in sufficient amounts of procaspase-8/c-FLIPL heterodimers at the DED filament, which supports the activation of caspase-8. In contrast, increased amounts of c-FLIPL 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>12.5% SDS gel</td>
			<td>self made</td>
			<td></td>
			<td>for two separating gels: 
			3.28 mL distilled H2O 
			2.5 mL Tris; pH 8.8; 1.5 M 
			4.06 mL acrylamide 
			100 &#181;L 10% SDS 
			100 &#181;L 10% APS 
			7.5 &#181;L 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 &#181;L 10% SDS 
			25 &#181;L 10% APS 
			7.5 &#181;L TEMED</td>
		</tr>
		<tr>
			<td>14.5 cm cell dishes</td>
			<td>Greiner</td>
			<td>639160</td>
			<td></td>
		</tr>
		<tr>
			<td>acrylamide</td>
			<td>Carl Roth</td>
			<td>A124.1</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-actin Ab</td>
			<td>Sigma Aldrich</td>
			<td>A2103</td>
			<td>dilution: 1:4000 in PBST + 1:100 NaN3</td>
		</tr>
		<tr>
			<td>anti-APO-1 Ab</td>
			<td>provided in these experiments by Prof. P. Krammer or can be purchased by Enzo</td>
			<td>ALX-805-038-C100</td>
			<td>used only for immunoprecipitation</td>
		</tr>
		<tr>
			<td>anti-caspase-10 Ab</td>
			<td>Biozol</td>
			<td>MBL-M059-3</td>
			<td>dilution: 1:1000 in PBST + 1:100 NaN3</td>
		</tr>
		<tr>
			<td>anti-caspase-3 Ab</td>
			<td>cell signaling</td>
			<td>9662 S</td>
			<td>dilution: 1:2000 in PBST + 1:100 NaN3</td>
		</tr>
		<tr>
			<td>anti-caspase-8 Ab C15</td>
			<td>provided in these experiments by Prof. P. Krammer or can be purchased by ENZO</td>
			<td>ALX-804-242-C100</td>
			<td>dilution: 1:20 in PBST + 1:100 NaN3</td>
		</tr>
		<tr>
			<td>anti-CD95 Ab</td>
			<td>Santa Cruz</td>
			<td>sc-715</td>
			<td>dilution: 1:2000 in PBST + 1:100 NaN3</td>
		</tr>
		<tr>
			<td>anti-c-FLIP NF6 Ab</td>
			<td>provided in these experiments by Prof. P. Krammer or can be purchased by ENZO</td>
			<td>ALX-804-961-0100</td>
			<td>dilution: 1:10 in PBST + 1:100 NaN3</td>
		</tr>
		<tr>
			<td>anti-FADD 1C4 Ab</td>
			<td>provided in these experiments by Prof. P. Krammer or can be purchased by ENZO</td>
			<td>ADI-AAM-212-E</td>
			<td>dilution: 1:10 in PBST + 1:100 NaN3</td>
		</tr>
		<tr>
			<td>anti-PARP Ab</td>
			<td>cell signaling</td>
			<td>9542</td>
			<td>dilution: 1:1000 in PBST + 1:100 NaN3</td>
		</tr>
		<tr>
			<td>APS</td>
			<td>Carl Roth</td>
			<td>9592.3</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Carl Roth</td>
			<td>4227.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford solution 
			Protein Assay Dye Reagent Concentrate 450ml</td>
			<td>Bio Rad</td>
			<td>500-0006</td>
			<td>used according to manufacturer&#39;s instructions</td>
		</tr>
		<tr>
			<td>CD95L</td>
			<td>provided in these experiments by Prof. P. Krammer or can be purchased by ENZO</td>
			<td>ALX-522-020-C005</td>
			<td></td>
		</tr>
		<tr>
			<td>chemoluminescence detector 
			Chem Doc XRS+</td>
			<td>Bio Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>cOmplete Protese Inhibitor Cocktail (PIC)</td>
			<td>Sigma Aldrich</td>
			<td>11 836 145 001</td>
			<td>prepared according to manufacturer&#39;s instructions</td>
		</tr>
		<tr>
			<td>DPBS (10x) w/o Ca, Mg</td>
			<td>PAN Biotech</td>
			<td>P04-53500</td>
			<td>dilution 1:10 with H2O, storage in the fridge</td>
		</tr>
		<tr>
			<td>eletrophoresis buffer</td>
			<td>self made</td>
			<td></td>
			<td>10x electrophoresis buffer: 
			60.6 g Tris 
			288 g glycine 
			20 g SDS 
			ad 2 L H2O 
			1:10 dilution before usage</td>
		</tr>
		<tr>
			<td>glycine</td>
			<td>Carl Roth</td>
			<td>3908.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgG1 HRP</td>
			<td>SouthernBiotech</td>
			<td>1070-05</td>
			<td>dilution 1:10.000 in PBST + 5% milk</td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgG2b</td>
			<td>SouthernBiotech</td>
			<td>1090-05</td>
			<td>dilution 1:10.000 in PBST + 5% milk</td>
		</tr>
		<tr>
			<td>Goat Anti-Rabbit IgG-HRP</td>
			<td>SouthernBiotech</td>
			<td>4030-05</td>
			<td>dilution 1:10.000 in PBST + 5% milk</td>
		</tr>
		<tr>
			<td>Interleukin-2 Human(hIL-2)</td>
			<td>Merckgroup/ Roche</td>
			<td>11011456001</td>
			<td>for activation of T cells</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Carl Roth</td>
			<td>6781.2</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Carl Roth</td>
			<td>3904.1</td>
			<td></td>
		</tr>
		<tr>
			<td>loading buffer 
			4x Laemmli Sample Buffer,10 mL</td>
			<td>Bio Rad</td>
			<td>161-0747</td>
			<td>prepared according to manufacturer&#39;s instructions</td>
		</tr>
		<tr>
			<td>Luminata Forte Western HRP substrate</td>
			<td>Millipore</td>
			<td>WBLUFO500</td>
			<td></td>
		</tr>
		<tr>
			<td>lysis buffer</td>
			<td>self made</td>
			<td></td>
			<td>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</td>
		</tr>
		<tr>
			<td>medium for adhaerent cells DMEM F12 (1:1) w stable Glutamine,&#160; 2,438 g/L</td>
			<td>PAN Biotech</td>
			<td>P04-41154</td>
			<td>adding 10% FCS, 1% Penicillin-Streptomycin and 0.0001% Puromycin to the medium</td>
		</tr>
		<tr>
			<td>medium for primary T cells</td>
			<td>gibco by Life Technologie</td>
			<td>21875034</td>
			<td>adding 10% FCS and 1% Penicillin-Streptomycin to the medium</td>
		</tr>
		<tr>
			<td>milk powder</td>
			<td>Carl Roth</td>
			<td>T145.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Carl Roth</td>
			<td>P030.3</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Carl Roth</td>
			<td>3957.2</td>
			<td></td>
		</tr>
		<tr>
			<td>PBST</td>
			<td>self made</td>
			<td></td>
			<td>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</td>
		</tr>
		<tr>
			<td>PBST + 5% milk</td>
			<td>self made</td>
			<td></td>
			<td>50 g milk powder + 1 L PBST</td>
		</tr>
		<tr>
			<td>PHA</td>
			<td>Thermo Fisher Scientific</td>
			<td>R30852801</td>
			<td>for actavation of T ells</td>
		</tr>
		<tr>
			<td>Power Pac HC</td>
			<td>Bio Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Precision Plus Protein Standard All Blue</td>
			<td>Bio Rad</td>
			<td>161-0373</td>
			<td>use between 3-5 &#181;L</td>
		</tr>
		<tr>
			<td>Protein A Sepharose CL-4B beads</td>
			<td>Novodirect/ Th.Geyer</td>
			<td>GE 17-0780-01</td>
			<td>affinity resin beads prepared according to manufacturer&#39;s instructions</td>
		</tr>
		<tr>
			<td>scraper</td>
			<td>VWR</td>
			<td>734-2602</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Carl Roth</td>
			<td>4360.2</td>
			<td></td>
		</tr>
		<tr>
			<td>shaker</td>
			<td>Heidolph</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sodium azide</td>
			<td>Carl Roth</td>
			<td>K305.1</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Carl Roth</td>
			<td>2367.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans Blot Turbo mini-size transfer stacks</td>
			<td>Bio Rad</td>
			<td>170-4270</td>
			<td>used according to manufacturer&#39;s instructions</td>
		</tr>
		<tr>
			<td>TransBlot Turbo 5x Transfer Buffer</td>
			<td>Bio Rad</td>
			<td>10026938</td>
			<td>prepared according to manufacturer&#39;s instructions</td>
		</tr>
		<tr>
			<td>TransBlot Turbo Mini-size nictrocellulose membrane</td>
			<td>Bio Rad</td>
			<td>170-4270</td>
			<td>used according to manufacturer&#39;s instructions</td>
		</tr>
		<tr>
			<td>Trans-Blot-Turbo</td>
			<td>Bio Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Chem Solute</td>
			<td>8,08,51,000</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Carl Roth</td>
			<td>3051.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Pan Reac Appli Chem</td>
			<td>A4974,1000</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring composition of cd95 death-inducing signaling complex and processing of procaspase-8 in this complex

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)-1&#946;-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.

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 ...

Watch this Scientific Journal Video about Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex at JoVE.com

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

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.

Extrinsic apoptosis is mediated by the activation of death receptors (DRs) such as CD95/Fas/APO-1 or tumor necrosis factor-related apoptosis-inducing ...

Research

JoVE Journal

Biochemistry

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Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. ...

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological ...

Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding

G-Protein-Coupled Receptors (GPCRs) are a large family of transmembrane receptors that play critical roles in normal cellular physiology and constitute a ...

A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between ...

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

Strategic Screening and Characterization of the Visual GPCR-mini-G Protein Signaling Complex for Successful Crystallization

The key to determining crystal structures of membrane protein complexes is the quality of the sample prior to crystallization. In particular, the choice ...

Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We ...

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Classic depletion-reconstitution experiments indicate that galectin-3 is a required splicing factor in nuclear extracts. The mechanism of incorporation of ...