Fig. 4.

Expression of WT RabGEF1 in Rabgef1^−/− BMCMCs normalizes c-Kit internalization to the levels seen in +/+ BMCMCs. (A) Rabgef1^−/− and +/+ BMCMCs were infected with viral supernatants from 293T cells transfected with control lentiviral vector (empty) or the lentiviral vector containing WT RabGEF1 cDNA, then sorted by GFP expression. RabGEF1 protein levels were analyzed by Western blot. Blots were reprobed with anti-GAPDH Abs to show loading. (B) Infected BMCMCs generated as in A were starved for 16 h in DMEM plus 10% FCS, then stimulated with 100 ng/ml SCF for the indicated times, and surface c-Kit expression was analyzed by flow cytometry. Gray, isotype control. Results in A and B are representative of those obtained in five separate experiments. (C) The bar graph represents the mean ± SEM of percent c-Kit internalization determinations from five separate batches of BMCMCs infected with lentiviral vectors. Percent c-Kit internalization was calculated by subtracting mean fluorescence intensity at 1 or 3 h from mean fluorescence intensity at 0 h and dividing this number by mean fluorescence intensity at 0 h (×100%). ∗, P < 0.05 vs. the indicated population. n.s., not significant.