Fig. 2. Cos2 associates with vesicles and plasma membrane.
A, total membranes prepared from S2 cells were washed in 0.15 m NaCl, then resuspended in 1.4 m sucrose, and overlaid with 1.22 m sucrose and then with 0.1 m sucrose. Following equilibrium density centrifugation (112,000 × g for 18 h), fractions were taken from the top of the tube. Each fraction was separated by SDS-PAGE and immunoblotted. Approximately 30% of Kinesin associates with vesicular membranes and is used here as a vesicular membrane marker (fractions 6–10) (61, 63). Fasciclin is a glycosylphosphatidylinositol-anchored protein and is used here as a plasma membrane marker (fractions 2–4) (66). The apparent accumulation of Fu in the plasma membrane to a greater degree than Cos2 is not consistently observed. B, S2 cells were transfected with HA-Cos2, then fixed, and stained with anti-HA-Alexa 488 and with 4,6-diamidino-2-phenylindole (not shown) to visualize DNA followed by confocal microscopy.