FIGURE 1.

CIN85 translocates to DRM-Hs of the plasma membrane following FcγRIIa cross-linking. Neutrophils (40 × 10⁶ cells/ml) were preincubated with 1 mm DFP for 10 min before FcγRIIa cross-linking at room temperature. Plasma membranes were prepared as described under “Experimental Procedures.” A, aliquots of total cell lysates, plasma membranes, and cytosol fractions from unstimulated cells were analyzed by immunoblots for the presence of CIN85. The loading of each cell fraction corresponded to equal cell equivalents. The same blot was reprobed for the plasma membrane marker flotillin-1. B, plasma membranes from resting or FcγRIIa-cross-linked cells were analyzed by immunoblotting for the presence of CIN85. The same blot was reprobed for flotillin-1, the protein loading control. A densitometric representation of the means of four independent experiments is shown in the histogram. C, plasma membranes of resting or FcγRIIa cross-linked (30 s) neutrophils were prepared as described under “Experimental Procedures” and solubilized in 1% Nonidet P-40. These samples were subject to ultracentrifugation on OptiPrep density gradients as described under “Experimental Procedures.” Thirteen gradient fractions were collected, and the proteins were precipitated and analyzed by immunoblotting with anti-CIN85 or anti-FcγRIIa antibodies. These data are representative of three independent experiments. WB, Western blot. Error bars, S.E.