Figure 4.

siRNA-knockdown of β-TrCP2 abrogates the Vpu-mediated loss of CD4 from the cell surface. 293TCD4 cells were transfected twice with the siRNAs targeting the indicated β-TrCP mRNAs or with a control siRNA and during the second transfection, plasmids encoding either a Vpu.GFP fusion protein or GFP alone were added. Two days post-transfection, cell-surface levels of CD4 were quantified in relation to GFP expression by flow cytometry, as reported for Figure 1E-G. β-TrCP mRNA levels were quantified by real-time PCR at the end of the experiment. β-TrCP mRNA values for control siRNA-transfected cells were set to 100%, and relative remaining levels of the specific mRNAs in knockdown cells were calculated by the 2^-ΔΔCt method. Shown are the arithmetic means ± SD of triplicates. Three independent experiments were performed.