Figure 1.
Purified PKC-ζ phosphorylates IRS-1, -3, and -4 but not IRS-2 in vitro. Kinase assays were performed using purified PKC-ζ as the kinase, [γ-32P]ATP, and recombinant IRS isoforms immunoprecipitated from cell lysates of transfected COS-7 cells as substrates. Top and middle panels, Autoradiograms demonstrating phosphorylated IRS isoforms and autophosphorylation of PKC-ζ, respectively, where present. Bottom panels, Immunoblotting controls confirming the presence or absence of IRS substrates in each of the in vitro kinase reactions. Representative autoradiograms and immunoblots are shown for experiments that were repeated independently five times. A, HA-tagged IRS-1 immunoprecipitated using an anti-HA antibody. B, IRS-2 immunoprecipitated with anti-IRS-2 antibody. C, Myc-tagged IRS-3 immunoprecipitated using an anti-myc antibody. D, Myc-tagged IRS-4 immunoprecipitated using an anti-myc antibody.