Figure 1
Figure 1. Expression of WT RabGEF1 in −/− BMCMCs normalizes basal FcϵRI surface expression, FcϵRI internalization, and IgE + Ag–induced degranulation and IL-6 production. (A) RabGEF1 +/+ and −/− BMCMCs were infected with control lentiviral vector (empty) or the lentiviral vector containing RabGEF1 cDNA (WT), then sorted by GFP expression. Total cell lysates were analyzed by Western blot using α-HA and α-RabGEF1 Abs. Blots were reprobed with α-GAPDH Abs to show loading. (B) Basal FcϵRI surface expression was analyzed on the indicated mast cell populations by flow cytometry. Data represent mean fluorescent intensity (MFI) of the indicated BMCMC population divided by MFI of the corresponding −/− empty vector control–treated BMCMCs (× 100%) pooled from 4 separate experiments + SEM (n = 4). (C) Infected BMCMCs generated as in panel A were sensitized for 16 hours in DMEM + 10% FCS containing 2 μg/mL IgE, then stimulated with biotinylated α-IgE Abs for the indicated times. Surface α-IgE was assessed by streptavidin (SA)–APC fluorescence and analyzed by flow cytometry. Gray represents SA-APC only. Histograms (top panels) are representative of results obtained in 5 separate experiments. The bar graph (bottom panel) shows the mean + SEM of percentage FcϵRI internalization determinations from 5 separate batches of BMCMCs. Percentage FcϵRI internalization was calculated by subtracting mean fluorescence intensity at 30 or 60 minutes from mean fluorescence intensity at 0 minute and dividing this number by mean fluorescence intensity at 0 minute (× 100%). (D-E) Infected BMCMCs generated as in panel A were sensitized for 16 hours with 2 μg/mL IgE, then (D) degranulation was quantified by measuring release of β-hexosaminidase (β-Hex) 1 hour after challenge with the indicated concentrations of DNP, and (E) IL-6 production was quantified 6 hours after challenge with 20 ng/mL DNP. Results in panels D-E are the mean + SEM of 9 separate determinations from 3 batches of BMCMCs. (B) * indicates P < .05 versus assigned −/− value of 100%. (C-E) + indicates P < .05; ++, P < .01; +++, P < .001 versus corresponding +/+ “empty” values; * indicates P < .05; **, P < .01; ***, P < .001 versus corresponding −/− “empty” values.

Expression of WT RabGEF1 in −/− BMCMCs normalizes basal FcϵRI surface expression, FcϵRI internalization, and IgE + Ag–induced degranulation and IL-6 production. (A) RabGEF1 +/+ and −/− BMCMCs were infected with control lentiviral vector (empty) or the lentiviral vector containing RabGEF1 cDNA (WT), then sorted by GFP expression. Total cell lysates were analyzed by Western blot using α-HA and α-RabGEF1 Abs. Blots were reprobed with α-GAPDH Abs to show loading. (B) Basal FcϵRI surface expression was analyzed on the indicated mast cell populations by flow cytometry. Data represent mean fluorescent intensity (MFI) of the indicated BMCMC population divided by MFI of the corresponding −/− empty vector control–treated BMCMCs (× 100%) pooled from 4 separate experiments + SEM (n = 4). (C) Infected BMCMCs generated as in panel A were sensitized for 16 hours in DMEM + 10% FCS containing 2 μg/mL IgE, then stimulated with biotinylated α-IgE Abs for the indicated times. Surface α-IgE was assessed by streptavidin (SA)–APC fluorescence and analyzed by flow cytometry. Gray represents SA-APC only. Histograms (top panels) are representative of results obtained in 5 separate experiments. The bar graph (bottom panel) shows the mean + SEM of percentage FcϵRI internalization determinations from 5 separate batches of BMCMCs. Percentage FcϵRI internalization was calculated by subtracting mean fluorescence intensity at 30 or 60 minutes from mean fluorescence intensity at 0 minute and dividing this number by mean fluorescence intensity at 0 minute (× 100%). (D-E) Infected BMCMCs generated as in panel A were sensitized for 16 hours with 2 μg/mL IgE, then (D) degranulation was quantified by measuring release of β-hexosaminidase (β-Hex) 1 hour after challenge with the indicated concentrations of DNP, and (E) IL-6 production was quantified 6 hours after challenge with 20 ng/mL DNP. Results in panels D-E are the mean + SEM of 9 separate determinations from 3 batches of BMCMCs. (B) * indicates P < .05 versus assigned −/− value of 100%. (C-E) + indicates P < .05; ++, P < .01; +++, P < .001 versus corresponding +/+ “empty” values; * indicates P < .05; **, P < .01; ***, P < .001 versus corresponding −/− “empty” values.

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