Analysis of RBDl internalization in Lrp1-deficient cells.

<p>RAW cells were transfected or not (NT) with one of the two Lrp1-siRNAs, Lrp1.1 or Lrp1.7, or with an unrelated siRNA as negative control (NC). 48h after transfection, <i>Lrp1</i> mRNA was determined by real time qPCR (A), at the protein level (B) or the cells were challenged for RBDl uptake (C, D). A, <i>Lrp1</i> mRNA is reduced in Lrp1-siRNAs transfected cells. B, LRP1 is down-regulated at the protein level in Lrp1-siRNA transfected cells. Immunoprecipitation of the α chain was performed using 8G1 mAb. The α chain was detectable in NT and NC cells but not in Lrp1.1-siRNA cells. C, RBDl uptake by LRP1-deficient cells is reduced. The cells were incubated with RBDl for 20 or 45 min. Uptake of the protein was analyzed by immunoprecipitation followed by immunoblot with the anti-GST mAb. S, supernatant recovered from cell culture medium after incubation. Total protein contents were monitored by Western blotting with a β-actin antibody. The blot shown was representative of two independent transfections. D, Internalization of biotinylated RBDl into Lrp1-deficient cells is compromised. NC siRNA-transfected cells, Lrp1.1 and Lrp1.7 siRNA-transfected cells were incubated with biotinylated RBDl for 30 min at 37°C. Pictures depict streptavidin-fluorescein labeling (green) or anti-LRP1 5A6 mAb labeling (red) with TOTO nuclear counterstain. Bar, 10 µm. Time exposures were 130 ms for overall pictures.