Figure 4

Cas cleavage during MG132-induced apoptosis. (A) Cell lysates from Cas-/- (EV), Cas-FL and WT MEFs treated with 1 μM MG132 were used in immunoblot analysis to detect Cas cleavage using an anti-p130Cas monoclonal antibody. (B) Cas-FL MEFs were treated with 1 μM MG132 for different times, and Cas cleavage was detected by immunoblot as in A. (C) Cas-FL MEFs were treated with 1 μM MG132, 25 μM Z-VAD-FMK, or 20 nM staurosporine (STS) alone, or in combination as indicated, and Cas cleavage was detected as in A. (D and E) Pools of Cas-/- MEFs stably transfected with control vector, or wt Cas, or Cas mutant D416E, or D748E, or the double mutant D416/748E, were treated with 1 μM MG132, and cleavage of Cas (in D) and of PARP and caspase-3 (in E) was detected by immunoblot. Additionally, p21 expression was studied in E by immunoblot. (F and G) Apoptosis ELISA assay (in F) and MTS assay (in G) were used to examine cell viability of MG132-treated Cas -/- MEFs expressing the constructs indicated.