Figure 1. The Ptpn11^E76K mutation enhances cytokine-induced PI3K/Akt/mTOR signaling.

(A) BM progenitors transduced with WT Shp2, Shp2 D61Y, Shp2 E76K, or control vector were sorted and differentiated to macrophages, which were starved in serum and cytokine-free medium for 48 hours and then stimulated with GM-CSF (50 ng/mL) for as long as 60 minutes. Protein extracts were prepared and immunoprecipitated with anti-Gab2 antibody followed by anti-pY immunoblotting. Blots were stripped and reprobed with anti-Shp2 and then anti-Gab2 antibodies. (B) BM-derived macrophages were generated from Ptpn11^E76K/+/Mx1-Cre⁺ and Ptpn11^+/+/Mx1-Cre⁺ mice 6–8 weeks following pI-pC administration. These cells were starved and then stimulated with GM-CSF (50 ng/mL) for the indicated periods of time. Whole cell lysates were prepared. Levels of p-ERK, p-Jak2, and p-Stat5 were determined by immunoblotting analyses. Blots were striped and reprobed with anti-ERK, anti-Jak2, anti-Stat5, and anti-Shp2 antibodies to check for protein loading. (C) The cell lysates were also examined for levels of p-AKT, p-mTOR, p-S6, and p-4E-BP1 by immunoblotting analyses. Blots were stripped and reprobed with anti-AKT and anti-mTOR antibodies to check for protein loading. Experiments were repeated three times with similar results obtained in each.