Fig. 2.

Effects of specific c-Yes knockdown by RNAi on the kinetics of endocytosis of integral membrane proteins at the Sertoli cell BTB in vitro. A: when c-Yes was knocked down by ≥70%, no off-target effects in potentially related signaling pathways were detected, including the steady-state level of c-Src in which β-actin served as the protein loading control. Each bar is the mean ± SD of 3 independent experiments. B: results of protein endocytosis assay (top) in which cultures at specified time points of the assay were terminated for lysate preparation to assess the kinetics of protein endocytosis. Each data point (bottom) is the mean ± SD of n = 4 independent experiments. *P < 0.05; **P < 0.01. C: effect of c-Yes silencing that impeded the endocytosis of BTB integral membrane proteins JAM-A and CAR was corroborated in this dual-labeled immunofluorescence analysis in which cells transfected with nontargeting control duplexes, endocytosed JAM-A protein colocalized with EEA-1, and more JAM-A were found to be internalized and to colocalize with EEA-1. These micrographs are representative findings of an experiment that was repeated 3 times with similar results. Bar, 20 μm.