FIGURE 5.

Defective Bcl-xL phosphorylation after mutation specifically of Ser-62. A, KB-3 cell lines expressing equivalent amounts of wild-type (WT) or HA-Bcl-xL(S62A) were untreated or treated with 200 nm vinblastine for 24 h, and extracts were immunoblotted for Bcl-xL with unphosphorylated and phosphorylated (P) forms indicated. Immunoblotting for GAPDH served as a loading control. B, KB-3 cell lines expressing wild-type or HA-Bcl-xL(S62A) were untreated or treated with 200 nm vinblastine for 24 h, and extracts were immunoprecipitated with Bcl-xL antibody and subjected to immunoblotting for phosphoserine (p-Ser) or Bcl-xL as indicated. Note that phosphoserine is only observed after immunoprecipitation of WT Bcl-xL or Bcl-xL(T47A) after vinblastine treatment. Lanes 3, 8 and 11 represent precipitations conducted in the absence of primary antibody. Immunoblotting for IgG was used as an additional control. C, KB-3 cell lines expressing wild-type or HA-Bcl-xL(S62A) were untreated or treated with 200 nm paclitaxel (TAX), VBL, or vincristine (VCR) for 24 h and subjected to immunoblotting for Bcl-xL, with phosphorylated form indicted (P).