Figure 4.
Figure 4. A subset of gp135 is delivered to the basolateral membrane before transcytosis and delivery to the apical surface. (A) MDCK-S cells were incubated with apical SBG-Block to block apical surface gp135. BG-PEG12-biotin was added to the medium bathing the basolateral membranes of MDCK-S cells. After a 1-h incubation at 4°C, cells were washed and moved to 37°C for 0–30 min. Cells were washed again, stained with BG549 (red) at 4°C, and then fixed for immunofluorescence with ZO1 (blue) and stained with streptavidin-488 (green). Labeling in the red channel of the merged image corresponds to gp135 that was newly delivered directly to the apical membrane. Labeling in the green channel corresponds to gp135 that was inserted first into the basolateral membrane and then delivered subsequently to the apical surface. Arrows indicate periciliary rings of transcytosed protein labeled with streptavidin-488. (B) Mean line scan intensity of streptavidin488-labeled and BG549-labeled gp135 at 6 min, as in Fig. 2 B, demonstrates delivery of transcytotic streptavidin488-labeled gp135 to the periciliary ring. All values were normalized to highest mean value of BG549 intensity at 6 min (left) or to the highest mean value of streptavidin488 intensity (right). n = 12 cells/condition from two independent experiments. (C and D) Surface SNAP-gp135 was blocked with SBG-Block. After washing, BG-PEG12-biotin was added to the medium, bathing either the basolateral (BL) membrane only or the apical and basolateral (AP + BL) membranes. Cells were moved to 37°C for 0 or 30 min, washed, then fixed for immunofluorescence with ZO1 (blue) and stained with streptavidin-488 (green). (C) Representative maximum-intensity projections demonstrate relative quantities of transcytosed versus total newly delivered SNAP-gp135. (D) Quantification of C normalized to streptavidin-488 intensity at 30 min in cells treated with AP + BL BG-PEG12-biotin. Values represent mean ± SEM from 186 (0 min BL), 191 (30 min BL), 161 (0 min AP + BL), and 204 (30 min AP + BL) cells. **, P < 0.0001. Data are representative of four independent experiments. Bars, 10 µm.

A subset of gp135 is delivered to the basolateral membrane before transcytosis and delivery to the apical surface. (A) MDCK-S cells were incubated with apical SBG-Block to block apical surface gp135. BG-PEG12-biotin was added to the medium bathing the basolateral membranes of MDCK-S cells. After a 1-h incubation at 4°C, cells were washed and moved to 37°C for 0–30 min. Cells were washed again, stained with BG549 (red) at 4°C, and then fixed for immunofluorescence with ZO1 (blue) and stained with streptavidin-488 (green). Labeling in the red channel of the merged image corresponds to gp135 that was newly delivered directly to the apical membrane. Labeling in the green channel corresponds to gp135 that was inserted first into the basolateral membrane and then delivered subsequently to the apical surface. Arrows indicate periciliary rings of transcytosed protein labeled with streptavidin-488. (B) Mean line scan intensity of streptavidin488-labeled and BG549-labeled gp135 at 6 min, as in Fig. 2 B, demonstrates delivery of transcytotic streptavidin488-labeled gp135 to the periciliary ring. All values were normalized to highest mean value of BG549 intensity at 6 min (left) or to the highest mean value of streptavidin488 intensity (right). n = 12 cells/condition from two independent experiments. (C and D) Surface SNAP-gp135 was blocked with SBG-Block. After washing, BG-PEG12-biotin was added to the medium, bathing either the basolateral (BL) membrane only or the apical and basolateral (AP + BL) membranes. Cells were moved to 37°C for 0 or 30 min, washed, then fixed for immunofluorescence with ZO1 (blue) and stained with streptavidin-488 (green). (C) Representative maximum-intensity projections demonstrate relative quantities of transcytosed versus total newly delivered SNAP-gp135. (D) Quantification of C normalized to streptavidin-488 intensity at 30 min in cells treated with AP + BL BG-PEG12-biotin. Values represent mean ± SEM from 186 (0 min BL), 191 (30 min BL), 161 (0 min AP + BL), and 204 (30 min AP + BL) cells. **, P < 0.0001. Data are representative of four independent experiments. Bars, 10 µm.

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