Analysis of internalization of biotinylated RBDl into RAW cells.

<p>A, Representative pictures obtained with streptavidin-fluorescein labeling. A membrane staining was pronounced when cells were incubated with RBDl for 1 h at 4°C. A cytoplasmic staining with punctuate appearance was observed when cells were incubated with RBDl for 30 min at 37°C. B,C, confocal pictures of individual cells depicting double staining with streptavidin-fluorescein and with either 8G1 mAb (B) or anti-EEA1 mAb (C) coupled withTRITC conjugate anti-mouse antibody. RBDl co-localized with LRP1 and with EEA1 in the cytoplasm. D, E, Immunofluorescence labeling of LRP1 with 8G1 mAb followed by Alexa-488 conjugate anti-mouse antibody on RAW cells either untreated (D) or incubated with α2M-MA (E) for 37°C at 30 min. A cytoplasmic staining with clustering appearance was observed when cells were incubated with α2M-MA. F, G, streptavidin-fluorescein labeling with TOTO nuclear counterstain of cells preincubated or not with GST-RAP for 30 min at 37°C before addition of biotinylated RBDl for 30 min at 37°C. The biotinylated RBDl was poorly internalized into the cells in the presence of GST-RAP. Pictures were taken with the same time exposure (137 ms). Bars, 5 µm.