Fig. 4. PKCδ regulates NADPH oxidase activity by phosphorylating p47^phox and inducing its translocation to the membrane fraction.

(A) Western blot analyses were performed with total cell lysates of PC-3 tumors treated with TAT or 38 mg/kg/day of ψδRACK for 4 weeks and immunoblotted with antibodies against phospho-serine/threonine (Ser/Thr) (*; p<0.05, n=3 each, Figure 4A). GADPH was used as a loading control. (B) Tumor lysates were immunoprecipitated with antibodies against p47^phox and probed for PKCδ. (*; p<0.05, n=3 each, Figure 4B). p47^phox was used as a loading control. (C) Phosphorylation levels of the co-immunoprecipitated p47^phox were checked by probing with anti-Ser/Thr antibodies (*; p<0.05, n=3 each, Figure 4C). PKCδ was used as a loading control. (D) Finally, tumor lysates were fractionated (as described in the methods) and the translocation of p47^phox from the cytosolic to the membrane (particulate) fraction was determined by probing each fraction with anti-p47^phox antibodies (Figure 4D, *; p<0.05, n=3 each). GADPH or Gαi was used as a loading control (IB: immunoblot).