FIGURE 9.

Mutational analyses demonstrating the crucial role of Ser-318 in CG-5-mediated HuR cytoplasmic translocation and degradation in LNCaP cells. A, effect of alanine substitution at Ser-158, Ser-221, and Ser-318 on CG-5-mediated HuR degradation. B, coimmunoprecipitation analysis of the effect of the Ser-318 → Ala substitution on HuR binding to PKCα. LNCaP cells ectopically expressing FLAG-tagged WT or S318A mutant form of HuR were treated with 5 μm CG-5 for 24 h. Immunoprecipitation (IP) with anti-FLAG-agarose conjugates and immunoblotting (WB) with anti- PKCα and anti-FLAG antibodies were performed as described in Fig. 6. C, immunocytochemical analysis of the cellular distribution and turnover of FLAG-tagged WT versus the S318A mutant of HuR in CG-5-treated cells. Scale bars = 10 μm.