In vivo association of c-Kit and PTP-RO in transfected COS-7 cells. COS-7 cells were cotransfected with c-Kit and PTP-RO cDNAs or c-Kit cDNA and control vector together with pcDNA3.1(−)/Myc-His/LacZ. Cells were starved with DMEM containing 0.5% FCS for 15 hours, followed by serum-free DMEM for 4 hours. SCF stimulation (500 ng/mL) was performed for the indicated time points and then cells were lysed with the modified RIPA buffer. After measuring β-galactosidase activity, clarified total cell lysates containing equal amounts of c-Kit were immunoprecipitated with anti–c-Kit antiserum (10 μL; Amgen). The precipitates were immunoblotted with the 4G10 antibody (1:3,000; A), anti-Flag antibody (2 μg/mL; Zymed; B), anti–c-Kit antibody (1 μg/mL; Santa Cruz; C), or anti–PTP-RO antibodies (D).