FIGURE 6.

Impact of Rabex-5 siRNAs-mediated knockdown or overexpression on L1 endocytosis. A, lysates of cells treated with Rabex-5 siRNAs or negative control siRNAs were analyzed by immunoblotting. B, cells cotransfected with Rabex-5 siRNAs and the indicated plasmids were incubated with L1-Ab, followed by immunocytochemistry. Arrowheads indicate Rabex-5-depleted cells. Scale bars represent 10 μm. C, fluorescence intensities of L1^WT-GFP, L1^K11R-GFP, and L1^Y1176A-GFP were quantified, and the percentage of perinuclear to total fluorescence was plotted. Data are presented as the mean ± S.E. (error bars) of triplicate experiments. ***, p < 0.001; n.s., not significant. D, N2a cells pretreated with CHX co-expressing of Myc-tagged Rabex-5 with indicated plasmids were surface-biotinylated on ice. Surface-biotinylated proteins were recovered with streptavidin-agarose from cell extracts and analyzed by immunoblotting. E, colocalization of Myc-tagged Rabex-5 (red) with L1^K11R-GFP (green) (upper panels) or L1^Y1176A-GFP (green) (lower panels). Scale bars represent 5 μm. F, fluorescence intensities were quantified, and the percentage of perinuclear to total fluorescence was plotted. ***, p < 0.001; n.s., not significant.