Figure 3.

Phosphorylation of CLOCK by Ca²⁺-dependent protein kinase C isoforms in vitro. (A) Serum-induced interaction between CLOCK and PKC. Cells were exposed to serum-rich medium for 30 min and then subjected to immunoprecipitation (IP) with CLOCK and PKC antibodies, followed by immunoblotting with both antibodies. (B) Histidine-tagged recombinant CLOCK was purified and subjected to in vitro PKC kinase assay in the absence or presence of 5 μM Gö6976. The reaction mixture was resolved by SDS–PAGE and transferred to a polyvinylidene fluoride membrane. The membrane was exposed to X-ray film for 24 h (upper panel). The same blot was then probed with CLOCK antibodies (lower panel). (C) Isoform specific kinase assays were carried out as described in (B). CTL, control; cPKC, classic PKC; PKC, protein kinase C; SDS–PAGE, sodium dodecyl sulphate–polyacrylamide gel electrophoresis; SS, serum shock.