J Clin Invest.
Transport kinetics and c-KIT signaling in BMMCs.
(A) Endocytosis of c-KIT. Smap1+/+ and Smap1–/– BMMCs were cultured, starved in the presence of cycloheximide, incubated with SCF at 37°C for the indicated times, and processed for flow cytometry analyses. The top panel displays the fluorescence intensity of c-KIT and cell numbers, whereas the bottom panel plots the percentages of internalized c-KIT calculated by considering the initial surface fluorescence to be 100%. BMMCs were prepared from 3 independent pairs of Smap1+/+ and Smap1–/– mice and processed for assays. Averages ± SD of internalized c-KIT were calculated for each incubation time (n = 3). (B) Immunofluorescence detection of c-KIT in BMMCs. The Smap1+/+ and Smap1–/– cells were incubated in the presence of SCF for the indicated times and stained for c-KIT. Scale bar: 10 μm. (C) Activation status of c-KIT signaling molecules. Wild-type and Smap1–/– BMMCs were incubated with SCF for the indicated times, and protein lysates were prepared and processed for immunoprecipitation/immunoblot analyses. Band densities were quantified, and averages ± SD are shown (n = 3). p-c-KIT, phosphorylated form of c-KIT; p-ERK1/2, phosphorylated form of ERK1/2; ub-c-KIT, ubiquitinylated c-KIT; c-KIT-associated Grb2, Grb2 recruited into anti–c-KIT immunoprecipitates. (D) DNA synthesis in BMMCs. Triplicate cultures of cells were prepared from each of the wild-type and Smap1–/– mice, incubated in the presence of IL-3 and/or SCF for 16 hours, and then treated with 3H-thymidine for 8 hours. The incorporation of 3H-thymidine into acid-insoluble fractions was measured, and averages ± SD are shown (n = 3). *P < 0.05.