Effect of inducible WT and R459M SHP-1 expression on hematopoietic differentiation of ES cells in OP-9 coculture. (A) ES cell transfectants were placed in coculture with OP-9 stromal cells in the presence of the indicated doses of Tet or in the absence of Tet. Lysates were prepared at the end of the first (day 5) and second (day 10) phases and were separated by SDS-PAGE on duplicate gels. Immunoblotting was performed first with anti–SHP-1 or anti-myc tag antibodies. The anti–SHP-1 blot was stripped and subsequently blotted with anti–SHP-2 and then anti-ERK1 antibodies. Anti–SHP-2 and ERK-1 blots indicate that the 10, 50, and 500 ng/mL Tet day 10 samples had more protein loaded than all the day 5 and day 10 no Tet samples. (B-D) ES cell transfectants were placed in coculture with OP-9 stromal cells in the presence or absence of 500 ng/mL Tet for the duration of the coculture and HCA. Representative results are shown 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) Average data from experiments using 2 independent WT (n = 4; ▦) 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 has been calculated and the average is shown, with standard errors indicated. Data were subjected to paired Student t test (^***P < .005; ^**P < .05; ^*P < .1).