ARH partially rescues saturated LDLR uptake. (A) BSC1-wt cells infected with adenovirus coding for CD8/LDLR were cultured in the presence of the indicated concentrations of tet and the rate of anti-CD8 antibody internalization relative to surface bound ligand was measured at 37°C, as described in Materials and methods. (B) Time course of single round internalization of prebound anti-CD8 antibody in BSC1 cells expressing EGFP-CLCa (○, •) or mCherry-ARH (□, ■). Shown are means (±SD) of at least three independent experiments. (C) BSC-wt cells or cells stably expressing LCa-EGFP, σ2-EGFP, ARH-wt-, ARH-F259A-, or ARH-252A255-mCherry were treated (+ µ2 k.d.) or not with µ2 siRNA to reduce AP2 levels, infected with CD8/LDLR adenovirus, and incubated with or without tet as indicated. The mean efficiency CD8/LDLR uptake as determined in at least two independent experiments is shown. Of note: to follow basal endocytic uptake of CD8, minimal exogenous expression of CD8/LDLR was necessary (i.e., overnight culture in the presence of 75 ng/ml tet). ***, confidence levels of P < 0.001 relative to wt control, 0 tet.