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Figure 4


Fig. 4. Western blot analysis of {alpha}-tubulin from apoptotic lysates. (A) HeLa cells (lanes 4-11) or Jurkat cells (lanes 15-22) were incubated with CTLs at increasing effector-to-target ratio (E:T) (0.5:1, 1:1 and 2:1) for 4 hours at 37°C. EGTA was included to inhibit granular-mediated killing (lanes 8-11, 19-22). Protein products were separated by 12% SDS-PAGE and analyzed by western blot with anti {alpha}-tubulin. Full-length {alpha}-tubulin is indicated by *, and cleaved {alpha}-tubulin by **. (B) Jurkat cells were incubated with CTLs at increasing E:T (2:1, 5:1 and 10:1) for 4 hours at 37°C. zVAD-fmk (100 µM final concentration) was included to inhibit caspase activation (lanes 5-8). Protein products were separated by 10% SDS-PAGE and analyzed by western blot with anti {alpha}-tubulin (upper panel) and anti-caspase 3 (lower panel). GrB-dependent processing of pro-caspase 3 is indicated by p20, and caspase-dependent processing is indicated by p19 and p17.





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