Figure 4

The SRCR domain is critical for the surface targeting and N-glycosylation of SR-AI. COS-7 cells were transfected with SR-AI and variants with truncated SRCR domains. A, Surface-targeted SR-AI variants were detected by live immunostaining (green). Cytosolic SR-AI variants were detected by immunocytochemistry (red). The yellow signal in the merged confocal images indicated that SR-AI, 371, and 341 were surface-targeted. Nuclei were counterstained with Hoechst 33258 (blue). Scale bar, 20 μm. B and C, Western blot analysis of total cell lysates and avidin pull-down of biotinylated lysates after PNGase F cleavage. β-actin and transferrin receptor (TfR) served as loading controls. D, Relative levels of surface-targeted SR-AI variants were quantified by densitometry. E, Western blot analysis of total cell lysates after PNGase F or Endo H cleavage. F and G, Transfected cells were incubated with fluorescent oAβ and AcLDL followed by immunostaining using anti-SR-A antibody. The experiments were repeated at least three times. Relative fluorescence intensities of internalized oAβ and AcLDL for more than 100 SR-AI-positive cells were quantified using MetaMorph software. Bars indicate mean ± SEM of three independent experiments. Experimental groups labeled with different letters (a, b, c) were significantly different from each other (p < 0.05).