Results: 4

1.
Fig. 3.

Fig. 3. From: RabGEF1 regulates stem cell factor/c-Kit-mediated signaling events and biological responses in mast cells.

SCF-induced c-Kit internalization is delayed in the absence of RabGEF1. (A) Rabgef1−/− and +/+ BMCMCs 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 levels were analyzed by flow cytometry. Gray, unstained control. (B) The bar graph represents the mean ± SEM of percent c-Kit internalization determinations from 6 (1 h) or 10 (3 h) separate experiments. 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 < 0.001 vs. +/+; +, P < 0.05 vs. corresponding 1-h value.

Janet Kalesnikoff, et al. Proc Natl Acad Sci U S A. 2006 February 21;103(8):2659-2664.
2.
Fig. 4.

Fig. 4. From: RabGEF1 regulates stem cell factor/c-Kit-mediated signaling events and biological responses in mast cells.

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.

Janet Kalesnikoff, et al. Proc Natl Acad Sci U S A. 2006 February 21;103(8):2659-2664.
3.
Fig. 1.

Fig. 1. From: RabGEF1 regulates stem cell factor/c-Kit-mediated signaling events and biological responses in mast cells.

SCF-mediated activation of Ras, Erk, and JNK is enhanced and prolonged, and cytokine production is enhanced, in Rabgef1−/− BMCMCs. (A) Cell surface expression levels of c-Kit in BMCMCs derived from Rabgef1−/− and +/+ mice were analyzed by flow cytometry. (B) Rabgef1−/− and +/+ BMCMCs were starved for 16 h in DMEM plus 10% FCS and then stimulated with 100 ng/ml SCF for the indicated times. Activated (Upper) and total (Lower) Ras levels were analyzed by Western blot using anti-Ras Abs. (C and D) Rabgef1−/− and +/+ BMCMCs were starved and stimulated as in B. Total cell lysates were subjected to Western blot analysis using anti-phospho-Erk1/2 (C) or anti-phospho-JNK (D) Abs (Left). The blots were reprobed with anti-Erk1/2 (C) or anti-GAPDH (D) Abs to show loading. (BD Right) Signals were quantified by densitometric scanning and corrected for loading. Results in AD are representative of results obtained in four or more separate experiments. (E) Rabgef1−/− and +/+ BMCMCs were stimulated as in B for 6 h, and IL-6 protein levels in the supernatants were quantified by ELISA. Data are mean ± SEM of the averages of duplicate determinations pooled from six (0 and 50 ng/ml SCF) or five (100 ng/ml SCF) separate experiments. ∗, P < 0.05; ∗∗, P < 0.01 vs. +/+; +, P < 0.05; +++, P < 0.001 vs. corresponding baseline (0 SCF) value. n.s., not significant.

Janet Kalesnikoff, et al. Proc Natl Acad Sci U S A. 2006 February 21;103(8):2659-2664.
4.
Fig. 2.

Fig. 2. From: RabGEF1 regulates stem cell factor/c-Kit-mediated signaling events and biological responses in mast cells.

SCF-induced phosphorylation of Akt is enhanced and prolonged in the absence of RabGEF1, and −/− BMCMCs exhibit a survival advantage, but reduced proliferation in response to SCF, compared with +/+ counterparts. (A) Rabgef1−/− and +/+ BMCMCs were starved for 16 h in DMEM plus 10% FCS and then stimulated with 100 ng/ml SCF for the indicated times. (Left) Total cell lysates were subjected to Western blot analysis using anti-phospho-Akt (Ser-473) Abs. The blot was reprobed with anti-GAPDH Abs to show loading. (Right) Signals were quantified by densitometric scanning and corrected for loading. (B) Rabgef1−/− and +/+ BMCMCs were cultured for 72 h in DMEM plus 10% FCS and then stained with Annexin V and analyzed by flow cytometry (Left). (Right) The bar graph represents mean ± SEM of pooled data from five separate experiments. (C) Rabgef1−/− and +/+ BMCMCs were starved for 16 h in DMEM plus 10% FCS, treated with the indicated concentrations of SCF for 48 h, and then labeled with 3H-thymidine for 6 h. Data represent mean ± SEM of triplicate determinations and are representative of results obtained in nine separate experiments (see text). (B and C) ∗, P < 0.05 vs. +/+; +, P < 0.05; +++, P < 0.001 vs. corresponding baseline (0 SCF) value. n.s., not significant. (D) Rabgef1−/− and +/+ BMCMCs were starved and stimulated as in A. (Upper) Total cell lysates were subjected to Western blot analysis using anti-p21 Abs, and the blot was reprobed with anti-GAPDH Abs to show loading. (Lower) Signals were quantified by densitometric scanning and corrected for loading. A and D are representative of results obtained in four and three separate experiments, respectively.

Janet Kalesnikoff, et al. Proc Natl Acad Sci U S A. 2006 February 21;103(8):2659-2664.

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