Figure 4
CHIP is involved in c-FLIP L degradation. (A), Calu-1 and H157 cells were treated with the indicated concentrations of 17-AAG for 48 h. (B), Calu-1 and H157 cells were treated with 1.0 μM 17-AAG for the indicated time. (C), Calu-1 and H157 cells were transfected with control siRNA (Crtli) and CHIP siRNA (CHIPi). 24 h after transfection, cells were reseeded and treated with 1.0 μM 17-AAG for 48 h. (D), Calu-1 cells were transfected with pcDNA-Myc-CHIP plasmid, and the cells were treated with 20 μM MG132 and 1.0 μM 17-AAG for 4 h. For immunoprecipitation, cell lysate was incubated with Myc antibody for 1 h and then incubated with protein A/G agarose(1:1 mix) at 4°C overnight(Left panel). Calu-1 cells were transfected with Lenti-Flag-c-FLIPL, and the cells were treated with 1.0 μM 17-AAG and 20 μM MG132 for 4 h. For immunoprecipitation, cell lysate was incubated with Anti-FLAG M2 beads at 4°C overnight (Right panel). The precipitated proteins were analyzed by Western Blot assay. (E), H157/c-FLIPL cells were transfected with plasmids Ub-HA and pcDNA-Myc-CHIP or pcDNA-Myc-CHIP(H260Q mutant), and then the cells were treated with 1.0 μM 17-AAG and 20 μM MG132 for 8 h. The cell lysates were subjected to immunoprecipitation with Flag antibody and subsequently Western Blot analysis.