Fig. 6. Sec8 associates with CASK and OSP/Claudin11 in OLs. (A) Sec8, CASK and OSP/Claudin11 coimmunoprecipitate. Mature OL lysates were incubated with anti-Sec8 Ab (2E12), anti-CASK Ab or pre-immune IgG and immunoprecipitates were analyzed by immunoblot. IP, immunoprecipitate; S, 10% of the supernatant fraction. (B) OSP/Claudin11, Sec8 and CASK cofractionate and float to low densities on sucrose gradients. The 1% Triton X-100 insoluble pellet fractions were isolated from mature OL lysates at 4°C, floated on sucrose-step gradients and analyzed by immunoblot; S, 15% of the soluble fraction. (C-E) Colocalization of (C) CASK (green) and OSP/Claudin11 (red), (D) Sec8 (green) and OSP/Claudin11 (red) or (E) Sec8 (green) and MOG (red) in OL processes was analyzed by confocal microscopy and ratiometric particle analysis for 680 randomly chosen puncta in 5-7 representative cells, three independent cultures. The Abs used for colocalization analysis were against the intracellular C-terminal regions of OSP/Claudin11 and MOG. Bars, 10 µm. Arrows indicate fluorescent puncta positive for OSP and CASK (C), positive for OSP and Sec8 (D), or positive for MOG, but not for Sec8 (E).