Figure 4

(A) GS induces dephosphorylation of pBAD. Whole cell lysates obtained from GS (50 μM) treated SCC4 cells were subjected to western blotting using specific antibodies for pBad, total Bad and α-tubulin. Panel shows the dephosphorylation of Bad in time-dependent manner (0 h - 12 h) while no difference was observed in the expression of total Bad protein in GS-treated SCC4 cells. α-tubulin served as loading control. (B) GS induces dissociation of Bad from 14-3-3ζ in head and neck cancer cells. Co-IP assays using specific antibodies were carried out and analyzed by western blotting as described in Materials and Methods. Treatment with GS induced dissociation of Bad from 14-3-3ζ as shown in panel (i) and (ii); association of Bad with Bcl2 as shown in panel (iii) and (iv); association of Bad with Bcl-xL (v) and (vi); and dissociation of pro-apoptotic protein Bax from Bcl-xL (vii and viii) as well as Bcl2 (ix and x) (C) Okadaic acid (OA) inhibits GS-induced dephosphorylation of Bad. Panel shows inhibition of dephosphorylation of Bad (Ser-136) and its dissociation from 14-3-3ζ in a time-dependent manner as determined using co-IP assays followed by western blotting.