FIGURE 5.

Knockdown of CD81 decreases the surface/total ratio of TfR2. Knockdown of CD81 decreases the surface/total ratio (S/T) of TfR2 but not TfR1. Hep3B-TfR2 cells were transfected with control siRNA (lane C and column C) or CD81 siRNA for 48 h. The cell surface proteins were biotinylated for 30 min on ice. The biotinylated proteins in 90% of the cell lysate were pulled down using streptavidin-agarose beads. 10% lysate was used as input. The samples were subjected to SDS-10% PAGE. The blot was probed with anti-TfR2, anti-TfR1, and anti-actin antibodies. The surface/total ratio of TfR2 and TfR1 was calculated. The fold change of the surface/total ratio of TfR2 and TfR1 in response to knockdown of CD81 was normalized to control siRNA-treated cells. The experiment was repeated four times with similar results.