Fig. 4. SH2-B mediates leptin-stimulated tyrosine phosphorylation of IRS1 and IRS2.

A, HEK293^LRb cells were transiently cotransfected with expression plasmids encoding IRS1 (1 μg) and SH2-Bβ, SH2-B(R555E), or ΔN504 (0.8 μg) as indicated. The cells were treated with 100 ng/ml leptin for 10 min. The cell extracts were immunoblotted (IB) with anti-phosphotyrosine. The positions of IRS1, SH2-Bβ, SH2-B(R555E), and ΔN504 were marked. CON, control. B, HEK293^LRb cells were transiently cotransfected with expression plasmids encoding IRS2 (1 μg) and SH2-Bβ (0.8 μg). The cells were treated with 100 ng/ml leptin for 10 min. The cell extracts were immunoblotted with αPY. The positions of IRS2 and SH2-Bβ were marked. C, HEK293^LRb cells were transiently cotransfected with expression plasmids encoding IRS2 (0.8 μg), Stat3 (0.4 μg), and Myc-tagged SH2-Bβ (0.3 μg) as indicated. The cells were treated with 200 ng/ml leptin for 10 min. The cell extracts were immunoblotted with anti-phosphotyrosine (αPY), anti-IRS2, anti-phospho-Stat3, anti-Stat3, and anti-Myc as indicated. The positions of IRS2, Stat3, and SH2-Bβ were marked. The phosphorylation of IRS2 and Stat3 was quantitated and normalized to total IRS2 or Stat3, respectively. D, SH2-B^−/−/LRb and SH2-B^+/+/LRb MEFs stably expressing LRb were treated with 100 ng/ml leptin for 10 min. The cell extracts were immunoprecipitated (IP) with αIRS1 and immunoblotted with anti-phosphotyrosine. The same blot was reprobed with αIRS1. E, SH2-B^−/−/LRb MEFs were infected with control (Con) or SH2-Bβ retroviruses, and stable clones were selected. The cell extracts were prepared from SH2-B^+/+/LRb and SH2-B^−/−/LRb MEFs infected with control or SH2-Bβ retroviruses, immunoprecipitated with αSH2-B, and immunoblotted with αSH2-B. F, control or SH2-Bβ retroviruses-infected SH2-B^−/−/LRb MEFs were treated with 100 ng/ml leptin for 10 min. The cell extracts were immunoprecipitated with αIRS1 and immunoblotted with αPY. The same blot was reprobed with αIRS1.