Apoptosis-induced shedding of the IL6R is caspase dependent, but PKC independent. (A) HepG2 cells expressing hIL6R were treated (6 hours) with CHX alone or in combination with αFas. The pan-caspase inhibitor zVAD-fmk (100 μM) was added as indicated. Western blot analysis of immunoprecipitated sIL6R was compared with an analysis of PARP (c-PARP, cleaved PARP), PKCδ (c-PKCδ, cleaved PKCδ), and β-actin. (B) HepG2 cells were treated as described in panel A. The inhibitor (each at 100 μM) for caspase 3 (zDEDV-fmk), caspase 8 (zIETD-fmk), and caspase 9 (zLEHD-fmk) was added 30 minutes prior to αFas stimulation. Western blot analysis was performed as before. (C) ELISA analysis of sIL6R production in the presence of caspase inhibitors. Values represent the mean (± SD) from 3 independent experiments. (D) HepG2 cells expressing hIL6R were incubated with PMA for 2 hours or preincubated with the pan-PKC inhibitor GF109203X (5 μM) or the inhibitor of Ca2+-dependent PKC Gö6976 (1 μM). Levels of immunoprecipitated sIL6R and cellular IL6R were determined by Western blot. (E) Comparative analysis of αFas-treated HepG2 cells in the presence of GF109203X (5 μM) and Gö6976 (1 μM).
