Fig. 2.

In Vitro Phosphorylation of IRS1 PH Domain on Ser24

A and B, wt (WT) or S24A (A) recombinant His6-IRS-PH domains were incubated with recombinant PKC isoforms or casein kinase II (CKII) as described in Materials and Methods. The kinase reactions were terminated and proteins separated by SDS PAGE followed by Western blot analysis with an antibody against pSer24. Parallel gels were also run and stained with either Coomassie (lower panels in A and B) or silver stain (SS in panel B) to confirm equal loading of kinase and recombinant IRS-PH domains. PKC-mediated anti-pSer24 immunoreactivity was ATP dependent (panel A) and Ser24 specific (panel B). C, In vitro kinase reactions were performed as described for panel A except that substrate used here was full-length rIRS1 (Myc-tagged) immunoprecipitated from serum-starved NIH/IR-rIRS1wt-MycHis6 cell lysates. Proteins were separated by SDS-PAGE followed by sequential Western blot analysis with antibodies against pSer24, pSer307, pSer612, or myc. The membranes were stripped clean of primary antibodies between each Western blot. Data are representative of at least three independent experiments. CKII, Casein kinase II.