In vivo association of c-Kit and PTP-RO in megakaryocytic cells. (A and B) CMK cells were starved in serum-free RPMI-1640 medium for 6 hours and incubated with or without 500 ng/mL SCF or FGF for 5 minutes at 37°C. Reaction was stopped by adding one-third volume of ice-cold PBS, followed by rapid centrifugation. After an additional wash with PBS, cells were lysed with the Triton X-100 lysis buffer. The clarified cell lysates were immunoprecipitated with either anti–c-Kit antiserum (Amgen; 10 μL), normal rabbit serum (NRS), or anti–FGF-R antiserum. The precipitates were immunoblotted with the 4G10 antibody (1:3,000; upper panel), anti–PTP-RO antibodies (R5702; 7 μg/mL; [A], middle panel; [B], lower panel), anti–c-Kit antibody (Santa Cruz, Santa Cruz, CA; 1 μg/mL; [A], lower panel) or anti–FGF-R antibody (Santa Cruz; [B], middle panel). (C and D) Mo7e cells were starved in RPMI-1640 medium containing 0.5% bovine serum albumin for 6 hours and stimulated with 500 ng/mL SCF for 10 minutes. Additional procedures were the same as in the case of CMK cells, except that cells were lysed with the modified RIPA buffer, and PY20 antibody (1:800) was used in (C) instead of the 4G10 antibody. Lysates were immunoprecipitated with anti–c-Kit or anti–PTP-RO antibodies (10 μL) or normal rabbit serum (NRS). The immunoprecipitates were immunoblotted with the 4G10 antibody (1:800), anti–PTP-RO antibodies (R5702), or anti–c-Kit antibodies, as indicated.