Fig. 5. PKC does not phosphorylate the Ser726 of -adducin. (A) GFP, GFP-PKC and GFP-PKC were transiently overexpressed in COS cells. Whole cell lysates were analyzed by immunoblotting with anti-phospho-adducin (Ser726), anti-adducin or anti-GFP. The Ser726 phosphorylation of adducin was measured and expressed as fold increase relative to the level in control GFP cells. (B) Endogenous PKC was immunoprecipitated by anti-PKC. The washed immunocomplexes were subjected to in vitro kinase assays using purified GFP--adducin (GFP-add) or its mutants as the substrate. The phosphorylation of GFP--adducin (wt) is defined as 100%. Values (means ± s.d.) are from three independent experiments. IVK, in vitro kinase assay.