FIGURE 3.

S6K1 stimulates the ubiquitination of IRS1 1–574 by CRL7. A, Coomassie stain analysis of the affinity-purified CRL7. An aliquot (∼3 pmol) of purified CRL7 was subject to SDS-PAGE, followed by Coomassie staining. The CRL7 components are indicated. B, phosphorylation of IRS-1 1–574 by S6K1. Phosphorylation reaction was carried out as described under “Experimental Procedures.” The reaction products were analyzed by immunoblot (IB) analysis using indicated antibodies. C, reconstitution of in vitro polyubiquitination of IRS-1 1–574 by CRL7. Polyubiquitination of IRS-1 1–574 was reconstituted, in the presence or absence of S6K1 treatment, as described under “Experimental Procedures.” Immunoblot analysis with indicated antibodies was performed to reveal the ubiquitination products and substrate input.