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. 2012 Oct 8;287(48):40758–40766. doi: 10.1074/jbc.M112.405209

FIGURE 5.

FIGURE 5.

Requirement of the IRS1 N terminus for efficient ubiquitination in vitro. A, insect cell-expressed IRS-1 261–574 is hyperphosphorylated. Dephosphorylation reaction was carried out as described under “Experimental Procedures.” The reaction products were analyzed by immunoblot analysis using anti-GST antibody. B, reconstitution of in vitro polyubiquitination of insect cell expressed IRS-1 261–574 by CRL7. Polyubiquitination of IRS-1 1–574 was reconstituted using glutathione bead enrichment assay as described under “Experimental Procedures.” Immunoblot (IB) analysis with indicated antibodies was performed to reveal the ubiquitination products and substrate input. C, comparison of ubiquitination of insect cell-produced IRS1–1-574 and IRS1–261-574. The time course reaction was carried out as described under “Experimental Procedures.”