Newly synthesized, not recycling, gp135 is delivered to the periciliary ring. (A and B) Newly delivered gp135 (green) in cells treated with CHX and/or with an extended recycling block with BG549. (A) Maximum-intensity projections of z-stacks representative of three independent experiments (ZO1, blue). Bar, 10 µm. (B) Mean line intensity of BG549 (old) and BG488 (newly delivered) across the apical membrane (0 min, dashed line; 10 min, solid line). At 10 min, untreated and recycling block-treated, but not CHX-treated, samples display peaks of heightened BG488 intensity proximal to the cilium. n = 15 cells/condition. 0-min BG488 values were normalized to the highest mean value of BG488 at 10 min for each condition. (C) Newly delivered, SNAP782-labeled gp135 was detected by Licor after SDS-PAGE. Control samples were not blocked before SNAP782 labeling. (D) Quantification of C normalized to SNAP782 intensity in untreated cells at 30 min. Values represent mean ± SEM of three (recycling block, gray; double block, white) or six (untreated, black) independent experiments. *, P < 0.05; **, P < 0.001; ***, P < 0.0001.