FIG. 13.

Intracellular oxidative stress activates the PKCδ/PKD/NF-κB pathway. (A) HeLa cells were stimulated with BSO (500 μM) for the indicated times. PKD was immunoprecipitated, and a substrate kinase assay was performed. PKD expression was revealed by immunoblotting. (B) HeLa cells were transfected with a pSUPER.PKCδ RNAi construct or the control vector. After 48 h, cells were stimulated with BSO (500 μM, 15 min). Endogenous PKD was immunoprecipitated (anti-PKD antibody) and analyzed for activation loop phosphorylation (anti-pS738/742 antibody). The blot was reprobed against PKD. Silencing of endogenous PKCδ was monitored by immunoblot analysis. (C) HeLa cells were transfected for 48 h with pSUPER (control), a pSUPER.PKCδ RNAi construct (PKCδ RNAi), or a pSUPER.PKD RNAi construct-pSuper.PKD2 RNAi construct combination (PKD RNAi). After 24 h, a second transfection with the NF-κB luc and β-gal reporters was performed. PKD and PKCδ expression was controlled by immunoblotting. All results are typical of three independent experiments.