PTPN11 mutants E76K, D61V, and D61Y confer constitutively elevated and prolonged GM-CSF–stimulated phospho-Erk levels in macrophage progenitors. (A) Representative Western blot showing 0- and 5-minute time points of GM-CSF stimulation of macrophage progenitor cultures; repeated in 3 independent experiments for MIEG3, WT, E76K, and D61V; repeated in 2 independent experiments for E61Y. (B) Band intensities were quantitated using densitometry, phospho-Erk band intensities were normalized to Erk band intensities, and data were compiled from all experiments and demonstrated graphically. Data shown as ± SEM; ^*P < .05 for E76K, D61V, and D61Y compared with MIEG3 at baseline. No statistical difference between MIEG3 and WT at baseline. (C) Western blot analysis demonstrating phospho-Erk levels following GM-CSF stimulation for 10 and 30 minutes demonstrating prolonged activation of Erk in macrophage progenitors transduced with each of the Shp-2 mutants. (D) A second independent experiment showing prolonged activation of Erk in macrophage progenitors transduced with each of the Shp-2 mutants for as long as 60 minutes following GM-CSF stimulation.