FIG. 3.

TNF-α induces rapid phosphorylation of p105 on serine 932. (A) NIH 3T3 cells were transiently cotransfected with expression vectors encoding wild-type (WT) HA-p105 or the indicated mutants and IKK2 or EV and treated with MG132 proteasome inhibitor for the last 4 h of a 48-h culture. HA-p105 was immunoprecipitated, and isolated protein was sequentially Western blotted with anti-phospho-Ser⁹³² (upper gel), anti-phospho-Ser⁹²⁷ (middle gel), and anti-p105C (lower gel) antibodies. Ip, immunoprecipitates. (B) HeLa cells were preincubated for 30 min with MG132 and then stimulated for the indicated times with TNF-α or control medium (C). Immunoprecipitates of endogenous p105 were then Western blotted sequentially with the indicated antibodies. (C) HeLa cells stably transfected with the indicated HA-p105 constructs were preincubated for 1 h with LLnL inhibitor and then stimulated for 15 min with TNF-α or control medium. Anti-HA immunoprecipitates were then sequentially Western blotted with the indicated antibodies.