Figure 5

CHIP determines ILK destabilization, ERK1/2/NF-κB inactivation, and cell growth inhibition after PI3K/HSP90 inactivation. AGS cells were transfected with shLuc or shHSP90 or pretreated with the HSP90 inhibitor 17-AAG (0.1 μM) for 48 h. Western blots show the expression of the indicated proteins (A), and NF-κB activation (B) and cell growth (C) were also determined. (D) MG132 pretreatment (0.5 h) reversed ILK expression and phosphorylated ERK1/2 at Tyr202/Thr204 (pERK1/2) in 17-AAG-treated AGS cells. (E) CHIP, cullin 4A, and cullin 4B were silenced in AGS cells by shRNAs. shRNA-transfected cells were treated with LY294002 or 17-AAG for 24 h. Western blotting showed the expression of ILK and phosphorylated ERK1/2 at Tyr202/Thr204 (pERK1/2) (F), and NF-κB activation (G) and cell growth (H) were also determined. (I) In shLuc- and shCHIP-transfected AGS cells treated with LY294002, ILK was immunoprecipitated, and its ubiquitylation was probed. For western blot analysis, β-actin was used as an internal control. For luciferase activity and cell growth, data are mean ± SD from three independent experiments. **P <0.01 and ***P <0.001 compared with control. ##P <0.01 and ###P <0.001 compared with shLuc or DMSO. NS: not significant. Upright triangle, increased expression; inverted triangle, decreased expression.