Figure 6
Rabaptin-5 deficiency diminishes mast cell IgE-dependent responses to specific antigen. (A) Control (shC) or Rabaptin-5 (shR) shRNA-treated BMCMCs were cultured with various concentrations of Alexa 647-labeled IgE (IgE-647) for the indicated times and then assessed for associated fluorescence. Top panel bar graph represents total associated fluorescence; bottom panel bar graph, fold increase in associated fluorescence compared with time 0 (incubated for 1 hour at 4°C). (B) shC- or shR-treated BMCMCs were cultured with low amounts of IgE-647 (25 ng/mL) in the presence (gray bars) or absence (black bars) of 100 μg/mL of brefeldin A, and associated fluorescence was assessed at the indicated times. +P < .05 for vehicle versus BFA; *P < .05 versus time 0 in the same treatment group. (C) C57BL/6-KitW−sh/W−sh mice were injected intraperitoneally with GFP+ shC- or shR-treated BMCMCs; 6 weeks later, peritoneal cells were harvested, stained with α-IgE, analyzed by flow cytometry, and MFIs of GFP+ cells were pooled to generate the bar graphs. (D) Ag-induced adhesion to fibronectin (FN) was assessed in shC- or shR-treated BMCMCs. Cells were sensitized with 1 μg/mL of DNP-specific IgE overnight, washed, and placed in FN-coated wells in the presence of the indicated concentrations of Ag and allowed to adhere for 1 hour. Data were pooled from triplicate determinations and are representative of the similar results that were obtained in each of the 5 experiments that were performed. (E) Migration to Ag through FN-coated transwells was assessed for shC- or shR-treated BMCMCs. Cells were prepared as in panel D, then placed in a transwell with the indicated concentrations of Ag and allowed to migrate for 6 hours. Migrated cells were counted by flow cytometry. Data were pooled from duplicate determinations and are representative of similar results that were obtained in the 3 experiments that we performed. (F) IL-6 produced from shC- or shR-treated BMCMCs, prepared as in panel D that were stimulated with the indicated concentrations of Ag for 6 hours; IL-6 was quantified by ELISA. (A,C-F) +P < .05, ++P < .01, +++P < .001 for shC- versus shR-treated cells.

Rabaptin-5 deficiency diminishes mast cell IgE-dependent responses to specific antigen. (A) Control (shC) or Rabaptin-5 (shR) shRNA-treated BMCMCs were cultured with various concentrations of Alexa 647-labeled IgE (IgE-647) for the indicated times and then assessed for associated fluorescence. Top panel bar graph represents total associated fluorescence; bottom panel bar graph, fold increase in associated fluorescence compared with time 0 (incubated for 1 hour at 4°C). (B) shC- or shR-treated BMCMCs were cultured with low amounts of IgE-647 (25 ng/mL) in the presence (gray bars) or absence (black bars) of 100 μg/mL of brefeldin A, and associated fluorescence was assessed at the indicated times. +P < .05 for vehicle versus BFA; *P < .05 versus time 0 in the same treatment group. (C) C57BL/6-KitW−sh/W−sh mice were injected intraperitoneally with GFP+ shC- or shR-treated BMCMCs; 6 weeks later, peritoneal cells were harvested, stained with α-IgE, analyzed by flow cytometry, and MFIs of GFP+ cells were pooled to generate the bar graphs. (D) Ag-induced adhesion to fibronectin (FN) was assessed in shC- or shR-treated BMCMCs. Cells were sensitized with 1 μg/mL of DNP-specific IgE overnight, washed, and placed in FN-coated wells in the presence of the indicated concentrations of Ag and allowed to adhere for 1 hour. Data were pooled from triplicate determinations and are representative of the similar results that were obtained in each of the 5 experiments that were performed. (E) Migration to Ag through FN-coated transwells was assessed for shC- or shR-treated BMCMCs. Cells were prepared as in panel D, then placed in a transwell with the indicated concentrations of Ag and allowed to migrate for 6 hours. Migrated cells were counted by flow cytometry. Data were pooled from duplicate determinations and are representative of similar results that were obtained in the 3 experiments that we performed. (F) IL-6 produced from shC- or shR-treated BMCMCs, prepared as in panel D that were stimulated with the indicated concentrations of Ag for 6 hours; IL-6 was quantified by ELISA. (A,C-F) +P < .05, ++P < .01, +++P < .001 for shC- versus shR-treated cells.

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