Fig. 2. CLIC4 is a downstream target of PKC in keratinocytes induced to differentiate by Ca²⁺. (A) Mouse keratinocytes were pre-treated with the indicated doses of PKC inhibitors for 1 hour, and then 0.5 mM Ca²⁺ was added in the medium for 24 hours prior to lysis and immunoblotting. (B) Mouse keratinocytes were infected with PKC DN Ad for 17 hours before the treatment with 0.5 mM Ca²⁺ for 24 hours. Actin was used as a loading control. Bis, bisindolylmaleimide I; Ro, rottlerin; Go, Go 6976. Similar data were obtained from two independent experiments. The numbers over the CLIC4 bands in A and B represent fold change determined by densitometry. (C) Purified recombinant CLIC1 or CLIC4 proteins were incubated with purified, kinase active recombinant PKC isoforms in appropriate reaction buffers with [-³²P]ATP, the reaction mixture was resolved by SDS-PAGE and then the phosphorylated proteins were visualized by phospho-imaging methods. Un, no recombinant PKC.