(A and C) Wild-type and Smap1–/– MEFs were transfected by EYFP-c-KIT, incubated with SCF for the indicated times, and processed for double-fluorescence detection of (A) c-KIT and lysotracker or (C) c-KIT and Hrs. (D) MEFs were incubated with dye-conjugated EGF for the indicated times and processed for double-fluorescence detection of EGF and lysotracker. In A, C, and D, the colocalization of the 2 molecules was analyzed and plotted as histograms for the indicated incubation period. Data are shown as averages ± SD (n = 50–70). Reproducible results were obtained for 2 independent Smap1–/– MEF cultures. *P < 0.05. (B) Double-immunofluorescence detection of endogenous SMAP1 and the indicated organelle marker in wild-type MEFs. The nuclei were stained by DAPI. The arrows in insets indicate colocalization of SMAP1 with Hrs or clathrin. Scale bars: 10 μm; 1 μm (insets).