Figure 7.

Rapid phosphorylation of GAP-A.p65 is not affected by overexpression of wild-type Sik or kinase-deficient Sik. Stable EMK cell lines containing the pLXSN vector alone (VECTOR) or expressing wild-type (SikWT) or kinase-deficient Sik (Sik 219K-M) were selected. (A) Expression of transfected Sik was detected by immunoblotting of total cellular proteins from the stable cell lines with anti-Sik antibody. (B) Sik kinase activity was measured by immunoprecipitation followed by in vitro kinase assays using total protein from EMK cell lines incubated for 1 h in high Ca²⁺ medium. Autophosphorylated Sik was detected by autoradiography. (C) Phosphorylation of GAP-A.p65 at 10 min and 1 h after Ca²⁺ addition to EMK cell lines was measured by immunoprecipitation with anti-GAP antibody followed by immunoblotting with anti-phosphotyrosine.