Fig. 3

IRS-1 and -2 tyrosine phosphorylation in islets isolated from Shb-transgenic mice (A), in RIN-neo and RIN-Shb cells (B), and in sorted GFP-RIN and GFP + Shb-RIN cells (C). (A) Islets (in groups of 1000) were isolated and cultured for 3–7 days before being incubated for 1 hr in the absence of serum. In two groups of islets, these were further stimulated for 10 min with 10% FCS or 10 µg/ml insulin as indicated. The islets were then washed, lyzed, and immunoprecipitated with IRS-1 or IRS-2 antibody prior to immunoblot analysis for phosphotyrosine (4G10) or IRS-2. (B) Control (RIN-Neo) or Shb (RIN-Shb) cells (10⁷) were maintained in the absence of serum for 1 hr or additionally stimulated with 10% FCS (S), 10 µg/ml insulin (Ins), or 100 ng/ml IGF-1 (Igf-1) for 10 min, before being washed in PBS, scraped, and lysed for immunoprecipitation using IRS-1 or IRS-2 antibodies as indicated. Aliquots of the lysates were taken prior to immunoprecipitation and analyzed. The immunoprecipitations were subjected to immunoblot analysis and probed for phosphotyrosine (4G10), IRS-1, IRS-2, FAK, and Shb immunoreactivity. (C) GFP-RIN or GFP + Shb-RIN cells (1–2 × 10⁵) were plated in culture dishes on day 0 (day of cell sorting and 1 day after lipofection). On day 1, cells were deprived of serum for 60 min, followed by addition of 100 ng/ml insulin for 10 min to some groups. The cells were then washed in PBS and lysed for immunoprecipitation using IRS-1 antibodies as indicated. The immunoprecipitates were subjected to immunoblot analysis and probed for phosphotyrosine (4G10).