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. 2011 May 2;286(26):23093–23101. doi: 10.1074/jbc.M111.233304

FIGURE 2.

FIGURE 2.

Cell-surface gC1qR is recruited to detergent-resistant lipid rafts after growth factor stimulation. A, rapidly proliferating nonconfluent A549 cells were surface-biotinylated with membrane-impermeable biotin for 2 h. Surface-biotinylated proteins were analyzed by immunoblotting with anti-gC1qR, integrin β1, and Pan-Ras antibodies. B, nonconfluent cells were serum-starved, subsequently treated with serum or growth factors as indicated in Fig. 1B, and surface-biotinylated. The surface-biotinylated proteins were analyzed by immunoblotting. WCL, whole cell lysate. C, detergent-resistant lipid rafts were isolated from rapidly proliferating nonconfluent cells. After sucrose gradient ultracentrifugation, the fractionated samples (from bottom to top) were analyzed by immunoblotting with anti-gC1qR, anti-insulin receptor β subunit (IRβ), anti-flotillin, and anti-cytochrome c antibodies. P indicates pellet. D, nonconfluent cells were serum-starved for 18 h and stimulated with serum or growth factors as indicated in Fig. 1B. Detergent-resistant lipid rafts were isolated from the cells and analyzed by immunoblotting with anti-CD44 and anti-gC1qR antibodies. GM1 was analyzed by blotting with biotin-labeled CTB and consecutive avidin-conjugated HRP.