Figure 6.
Effects of PKC‐ι siRNA on Bad phosphorylation and Bad/Bcl‐xL heterodimerization. (a) Both RWPE‐1 cells and DU‐145 cells were treated with PKC‐ι siRNA for 24 h and cell lysates (300 µg) were immuoprecipitated with Bad mouse monoclonal agarose‐conjugate (5 µg). The resulting supernatant and immunocomplex beads were subjected to SDS‐PAGE. The first column is the negative control containing normal mouse IgG agarose conjugate with cell lysate. Phosphorylation of Bad was analysed using phospho‐specific antibodies: Bad Ser‐112 (S112), Ser‐155 (S155) and Ser‐136(S136). Bound Bcl‐xL with Bad and unbound Bcl‐xL was also analysed. (b) Cell lysates of endogenous protein (1 mg) were immunoprecipitated with Bad and different level of phosphorylation status of Bad in normal RWPE‐1 cells and DU‐145 cells was also examined.