Subcellular localization of collectrin in kidney tissues and mIMCD3 cells.

<p><i><u>Panel a:</u></i> Western blot analyses of snapin, aquaporin-2 and collectrin, isolated from plasma membrane (PM) and vesicle membrane (VM) fractions. Collectrin antibody detected a single band of ∼40 kDa. <i><u>Panel b:</u></i> N-glycosidase (N) and O-glycosidase (O) treatment generates ∼30 kDa and ∼35 kDa respective bands. <i><u>Panel c:</u></i> Immunoreactivity of collectrin is detected by confocal microscopy; five layers (1 to 5) are shown and collectrin is seen in cytoplasm (2 to 5 cuts) and apical membrane (1 cut). <i><u>Panel d:</u></i> Apical view of mIMCD3 cells. Collectrin (green) and acetylated α-tubulin (red), a primary ciliary marker, are depicted. <i><u>Panel e:</u></i> Higher magnification shows that acetylated α-tubulin (red) and collectrin (green) co-localize. <i><u>Panel f:</u></i> Collectrin (green) is seen surrounding the γ-tubulin, a marker for the basal body (red). <i><u>Panel g:</u></i> Immunoelectron microscopy shows gold particles localized mainly in microvesicles around the basal body of the primary cilium (arrow heads), whereas few gold particles are observed at the peripheral region of proximal centriole. Scale bars, 20 µm for panels c, d, e, and f and 500 nm for panel g.