Socs7 affects insulin signaling and IRS1 protein stability. (A) Insulin stimulation of wild-type or Socs7 –/ – mice. Six-week-old female mice were injected with a 5-unit bolus of human insulin. Soleus muscle was harvested at the times indicated. IRS1 tyrosine phosphorylation (p-Tyr) was assayed by immunoprecipitation and immunoblot. The blots were stripped and reprobed for IRS1 to assess protein loading. Relative molecular weight markers are indicated in kDa. (B) Schematic representation of SOCS7 mutants. Wild-type SOCS7 contains 4 polyproline domains (P), an SH2 domain, and a SOCS box domain (SB). The ΔN mutant lacks the 4 N-terminal polyproline domains. The ΔSB mutant contains a deletion of the C-terminal SOCS box. The R→K mutation includes 2 point mutations in the phosphotyrosine-binding domain of the SH2 domain. (C) HEK293 cells were transfected with cDNAs for ubiquitin, Irs1, and wild-type or mutant Socs7 as indicated. Forty-eight hours after transfection, cells were harvested, and immunoprecipitation and immunoblotting for IRS1 were performed. (D) HEK293 cells were transfected as in C. Then, 42 hours after transfection, cells were preincubated for 30 minutes with either 20 μM of the proteasome inhibitor MG132 (right 3 lanes) or methanol carrier as a control. Cells were then stimulated with 100 nM insulin for 6 hours before harvest and immunoprecipitation with IRS1 and immunoblot with anti-ubiquitin antibody. Whole cell lysates were blotted with an antibody recognizing SOCS7. IRS1-Ub, ubiquitinated IRS1.