Fig. 3. SH2-B mediates a JAK2/SH2-B/IRS1 or IRS2 tertiary complex.

A, HEK293 cells were transiently cotransfected with expression plasmids encoding IRS1 (1 μg), JAK2 (0.8 μg), and SH2-Bβ (0.6 μg) as indicated. The cell extracts were prepared 48 h after transfection, immunoprecipitated (IP) with αJAK2, and immunoblotted (IB) sequentially with αIRS1, αSH2-B, and αJAK2. B, HEK293^LRb cells were transiently cotransfected with expression plasmids encoding IRS1 (1.2 μg), JAK2 (0.6 μg), and SH2-Bβ or ΔN504 (0.6 μg) as indicated. The cells were treated with 100 ng/ml leptin for 10 min. The cell extracts were immunoprecipitated with αJAK2 and immunoblotted with αIRS1. The same blot was reprobed with anti-phosphotyrosine (αPY). C, HEK293^LRb cells were transiently cotransfected with expression plasmids encoding IRS2 (1.2 μg), JAK2 (0.6 μg), and SH2-Bβ or ΔN504 (0.6 μg) as indicated. The cells were treated with 100 ng/ml leptin for 10 min. The cell extracts were immunoprecipitated with αJAK2 and immunoblotted with αIRS2. The same blot was reprobed with anti-phosphotyrosine.