Figure 5

N-Glycosylation processing modified the spatial arrangement of Kv3.1b protein-containing particles in subdomains of the cell membrane. Representative micrographs of glycosylated (WT) (A) and unglycosylated (N220/229Q) (B) Kv3.1b proteins expressed in NB_1(-Mgat1) (left panels) and control (right panels) cells acquired in TIRF (upper panels), DIC (middle panels) and wide-field (lower panels) modes. Representative scale bar (5 μM) was similar for all acquired images. The dashed circle denotes the cell body and the projections from the cell body represent the outgrowths. (C) Percent fluorescence in the outgrowth of parental and N-glycosylation mutant cells lines stably expressing glycosylated (WT) and unglycosylated (N220/229Q) Kv3.1b proteins. Data are presented as the mean ± SEM and were compared by one-way ANOVA (* p < 0.03). n represents the number of cells analyzed. (D) Number (top panel), area (middle panel) and mean intensity (bottom panel) of particles in cell body and outgrowth of NB_1(-Mgat1) and control (NB_1) cell lines expressing glycosylated (WT) and unglycosylated (N220/229Q) Kv3.1b proteins. Insets represent differences in the particle parameters due the type of N-glycan attached to the Kv3.1 protein in cell body and outgrowth. Graphs denote mean ± SEM and were compared by Student’s t-test (p < 0.05). (*) denotes a comparison between glycosylated and unglycosylated Kv3.1b in the same cell line and (*) represents a comparison between either glycosylated or unglycosylated Kv3.1b a cell line. n denotes the number of particles examined.