Journal: Journal of immunology (Baltimore, Md. : 1950)
Article Title: Effector CD4+ T Cells Generate Intermediate Caspase Activity and Cleavage of Caspase-8 Substrates
Figure Lengend Snippet: Activated T cells possess active full-length caspase-8, active cleaved caspase-3, full-length FLIP L , p43FLIP, and RIP1 complexed with active caspases. Freshly isolated polyclonal T cells ( lanes 1 and 3 ) or day 4 effectors ( lanes 2 , 4 , and 5 ) were incubated with control peptide z-FA-fmk ( lanes 1 - 4 ) or 100 μ M nonbiotinylated z-VAD ( lane 5 ) at 37°C for 15 min. All cells were then incubated with 10 μ M biotin-VAD-fmk for an additional 15 min at 37°C. Subsequently, cells were lysed in buffer containing 20 μ M biotin-VAD ( lanes 1 - 5 ). A portion of lysates were then incubated with avidin-Sepharose beads to precipitate active caspases. Immunoblot analysis was performed on precipitates for caspase-8 ( top panel ), caspase-3 ( middle panel ), or RIP1 ( bottom panel ) to reveal active caspases and RIP1 cleavage products. As a positive control for the presence and size of caspase-8, caspase-3, and RIP1, a portion of nonprecipitated whole cell lysates (WCLs) was included in the analysis from fresh ( lane 1 ) or day 4 effectors ( lane 2 ). B , Active caspases were precipitated from day 4 effector lysates and analyzed by immunoblot for the indicated molecules. WCLs were included as a reference comparison. A longer exposure of the caspase-8 immunoblot is included to illustrate precipitation of p43/41 caspase-8. C , Day 4 effectors were treated with FasL for 1 or 2 h, followed by biotin-VAD for 15 min. Active caspases were then precipitated and immunoblot analysis was performed for caspase-8. A portion of the fraction not binding to avidin-Sepharose (Flow Through) as well as WCLs were included in analysis. Bands were revealed using anti-caspase-8 Ab ( top panel ) or HRP-conjugated Strepavidin ( middle and bottom panels ). Bands at 55 and 43/41 kDa were identified, which corresponds with the bands revealed in the caspase-8 immunoblots ( top and middle panels ). In addition, a band of 17 kDa was observed that corresponds with cleaved p17 caspase-3 ( bottom panel ).
Article Snippet: Blots were washed three times for 15 min each and incubated for 90 min with 0.5 μ g/ml (caspase blots) or 1 μ g/ml (all others) of appropriate secondary Ab conjugated to HRP (Santa Cruz Biotechnology).
Techniques: Isolation, Incubation, Avidin-Biotin Assay, Positive Control, Binding Assay, Flow Cytometry, Western Blot