Effect of inducible WT and R459M SHP-1 expression on hematopoietic differentiation of ES cells in EBs. ES cell transfectants were placed in EB culture in the presence or absence of 500 ng/mL Tet for the duration of EB development and HCAs. (A) Lysates were prepared from EBs at the times shown and were immunoblotted with the anti-myc epitope tag antibody 9E10 (top row). The position of R459M SHP-1 is indicated. Blots were stripped and reprobed for anti–SHP-1 or anti–SHP-2 (bottom row). Representative results are shown for experiments performed using WT (B) and R459M (C) SHP-1–expressing transfectants. Numbers of GM/M, erythroid (E), GEMM, and secondary EB colonies obtained are shown. (D) Combined data from experiments using WT (n = 3; ▦) and R459M (n = 3; □) SHP-1–expressing transfectants are shown. For each experiment, the fold change in the number of each colony type obtained -Tet compared with +Tet was calculated, the average was taken, and standard errors are shown. Data were subjected to paired Student t test (^***P < .005; ^**P < .05; ^*P < .1).