FIGURE 2.
Internalization of toxin receptors. Internalization of reversibly biotinylated cell surface proteins into serum-cultured murine fibroblasts (MEFs) either left non-treated (A) or treated with TcdB (B) was induced by temperature shift to 37°C. Cells were collected at the indicated times. Cells were either left non-treated or exposed to GSH on ice to strip off biotin from still surface-exposed molecules. Biotinylated proteins were retrieved on neutravidin-agarose, eluted, and analyzed by immunoblotting. (C) Internalization of toxin receptors into non-treated fibroblasts was quantified by densitometry of immunoblot (n = 3). (D) Lysate from non-treated fibroblasts (lysate) and immunoprecipitate biotinylated cell surface proteins were subjected to immunoblotting and analyzed with the indicated CSPG4 antibodies. (E) Internalization of biotinylated cell surface proteins was further analyzed in serum-starved fibroblasts pretreated anisomycin (30 μM, E). Representative immunoblots are from one of three independent experiments.