SHP-1 acts at early and intermediate stages of differentiation. (A) WT and (B) R459M SHP-1 ES cell transfectants were differentiated according to the 3-phase coculture protocol (Figure 1A(i)). Cells were plated in the presence of 500 ng/mL Tet for the duration, or Tet was removed for 1 of the 3 phases. Culture conditions over the 3 phases are indicated. Numbers of colonies present in the HCAs (phase 3) were determined, and representative results are shown. □ indicates GM/M; ▦, E; ▨, GEMM; and ▪, EB. (C) R459M SHP-1 ES cell transfectants were placed in EB culture for 3 days in the presence or absence of 500 ng/mL Tet. EBs were dissociated, and cells (i) were plated directly into HCAs or (ii) were subjected to a second round of embryoid body formation, in the presence of Tet in both cases, before plating into HCAs. Numbers of colonies present in the HCAs were determined, and representative results are shown. (D) R459M clone 20 ES cells were plated in the presence (+) or absence (-) of 500 ng/mL Tet for 24 hours in the case of undifferentiated ES cells (ES) or for 72 hours in the case of primary embryoid body formation (EB). After these periods of time, cells were washed and 500 ng/mL Tet was added back to the ES cell liquid cultures for 24 and 48 hours (-/+). Primary embryoid bodies were dissociated and plated into a second round of embryoid body formation with the readdition of 500 ng/mL Tet. Lysates were prepared at the times indicated, and immunoblotting was performed with anti–SHP-1 antibodies first. For EB samples, the amount of protein obtained was too low to detect a clear SHP-1 signal. Blots were stripped and reprobed with anti-myc antibodies and then stripped and reprobed with anti-ERK1 antibody as a loading control. The position of proteins is indicated.