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. Author manuscript; available in PMC: 2009 Jan 31.
Published in final edited form as: Mol Cell. 2008 May 23;30(4):403–414. doi: 10.1016/j.molcel.2008.03.009

Figure 5. The CUL7 E3 Binds IRS-1 and Promotes Its Polyubiquitination In Vitro.

Figure 5

(A) Dephosphorylation of IRS-1. Purified insect-cell-produced human IRS-1 (60 ng) was incubated with λ phosphatase (1 unit; lane 3) or alkaline phosphatase (1 unit; lane 5) at 37°C for 30 min. The reaction products were analyzed by immunoblot analysis using anti-IRS-1 antibody.

(B) Phosphorylation of IRS-1 by S6K. Purified human IRS-1 (60 ng) was incubated with S6K (0.1, 1, and 10 pg for lanes 2–4, respectively) at 37°C for 30 min. The reaction products were analyzed by immunoblot analysis using antibodies recognizing IRS-1 phosphorylated at Ser307 (upper) or for total IRS-1.

(C) In vitro interaction between Fbw8 and IRS-1. The binding experiment was carried out as described in the Supplemental Data, with input (20%) shown in lane 1.

(D and E) Reconstitution of in vitro polyubiquitination of IRS-1 by the CUL7 E3 and kinetic analysis. Polyubiquitination of IRS-1 was reconstituted as described in the Experimental Procedures.