Figure 1
Rabaptin-5 knock-down does not impair canonical Rab5 processes in BMCMCs. (A) Total cell lysates from BMCMCs treated with control (shC) or Rabaptin-5 (shR) targeted shRNA constructs were resolved by SDS-PAGE and probed with the indicated primary antibodies. (B) BMCMCs generated as in panel A were processed for confocal microscopy as described in Document S1, (“Immunofluorescence”) and stained with α-EEA1 (red) and α-Rab5 (green) antibodies and phalloidin (blue) to identify the actin cytoskeleton. The regions outlined by dashed boxes are shown magnified, and as individual channels, above the overlay. (C) BMCMCs generated as in panel A were transiently transfected with the indicated GFP constructs by electroporation for 12 to 24 hours and processed for immunofluorescence as in panel B with GFP fluorescence in green and α-EEA1 in red. (D) Endosome sizes from individual shC- or shR-treated BMCMCs prepared as in panel C were measured as described in Document S1, “Immunofluorescence,” averaged, pooled from 3 separate experiments, and compared (***P < .001 vs corresponding “control” untransfected cells). (E) BMCMCs generated as in panel A were sensitized with IgE, stimulated with biotinylated α-IgE Abs for the indicated times, and surface α-IgE was assessed by flow cytometry. The bar graph shows the mean plus or minus SEM of percentage FcϵRI internalization determinations from 4 separate batches of BMCMCs (***P < .001 vs MFI at 30 minutes). (F) BMCMCs generated as in panel A were pulsed with 0.5 mg/mL of Cy 5-labeled BSA for 20 minutes, washed, and chased with unlabeled BSA. Associated fluorescence was measured at the indicated times after washout by flow cytometry. To prevent endosome acidification, some cells were pretreated with 50 μM of bafilomycin for 10 minutes before the BSA pulse. Graphs represent data transformed to show fold increases above unlabeled cells pooled from 3 pairs of shC- or shR-treated BMCMCs. Linear regression analysis was performed to generate lines of best fit with the slopes and 95% confidence intervals noted. Scale bars in panels B and C represent 7.5 μm.

Rabaptin-5 knock-down does not impair canonical Rab5 processes in BMCMCs. (A) Total cell lysates from BMCMCs treated with control (shC) or Rabaptin-5 (shR) targeted shRNA constructs were resolved by SDS-PAGE and probed with the indicated primary antibodies. (B) BMCMCs generated as in panel A were processed for confocal microscopy as described in Document S1, (“Immunofluorescence”) and stained with α-EEA1 (red) and α-Rab5 (green) antibodies and phalloidin (blue) to identify the actin cytoskeleton. The regions outlined by dashed boxes are shown magnified, and as individual channels, above the overlay. (C) BMCMCs generated as in panel A were transiently transfected with the indicated GFP constructs by electroporation for 12 to 24 hours and processed for immunofluorescence as in panel B with GFP fluorescence in green and α-EEA1 in red. (D) Endosome sizes from individual shC- or shR-treated BMCMCs prepared as in panel C were measured as described in Document S1, “Immunofluorescence,” averaged, pooled from 3 separate experiments, and compared (***P < .001 vs corresponding “control” untransfected cells). (E) BMCMCs generated as in panel A were sensitized with IgE, stimulated with biotinylated α-IgE Abs for the indicated times, and surface α-IgE was assessed by flow cytometry. The bar graph shows the mean plus or minus SEM of percentage FcϵRI internalization determinations from 4 separate batches of BMCMCs (***P < .001 vs MFI at 30 minutes). (F) BMCMCs generated as in panel A were pulsed with 0.5 mg/mL of Cy 5-labeled BSA for 20 minutes, washed, and chased with unlabeled BSA. Associated fluorescence was measured at the indicated times after washout by flow cytometry. To prevent endosome acidification, some cells were pretreated with 50 μM of bafilomycin for 10 minutes before the BSA pulse. Graphs represent data transformed to show fold increases above unlabeled cells pooled from 3 pairs of shC- or shR-treated BMCMCs. Linear regression analysis was performed to generate lines of best fit with the slopes and 95% confidence intervals noted. Scale bars in panels B and C represent 7.5 μm.

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