Dynamics of GEMs in WEHI-231 and CD45-deficient clone 10-5 cells. (A) Characterization of GEMs in WEHI-231 and 10-5 cells. Cells were either left unstimulated (–) or were stimulated for 5 minutes at 37°C with 20 μg/mL (Fab′)2 fragments of antimouse IgM Ab (+), after which they were lysed in 0.5% Triton X-100 and subjected to sucrose gradient ultracentrifugation. Equal volumes of gradient fraction were separated by SDS-PAGE and blotted with HRP-conjugated CTB subunit to detect the ganglioside GM1, or with Abs against paxillin and CD71. Fr indicates fraction. (B) Association of Lyn, IgM, Ig-α, and Csk with GEMs. WEHI-231 and 10-5 cells were either left unstimulated or were stimulated with 20 μg/mL antimouse IgM Ab for 1, 5, or 60 minutes, and then treated up to the ultracentrifugation step as in panel A. Equal volumes of GEMs (sucrose gradient fractions 4 and 5 combined) were separated by SDS-PAGE and blotted with Abs against Lyn, IgM, Ig-α, and Csk. (C) Percentages of GEM-associated Lyn (•), IgM (○), Ig-α (▪), and Csk (□) in WEHI-231 (solid line) and 10-5 (broken line) cells.
