Vpu-induced proteasomal degradation of tetherin involves ERAD.

<p>(A) The ERAD pathway is involved in Vpu-mediated tetherin depletion. 293T cells were transfected in duplicate with 100 nM of either a non-silencing control siRNA, or of a siRNA pool targeting p97. Twenty-four hours later, these cells were transfected with Flag-tagged tetherin in the presence or absence of Vpu (with a molar ratio of 2∶1 in favor of Vpu). Duplicate cell lysates were pooled for western blot analysis (left panel). Actin served as a loading control. Note that both parts of the figure come from the same scan of the same blot. The effect of the different siRNAs on Vpu-mediated tetherin depletion was quantified by densitometry and a plot was generated from the results of two independent experiments performed in duplicate (right panel). The values obtained in the absence of Vpu were given the arbitrary value of 100%. (B) Vpu-mediated tetherin depletion does not require ubiquitination of tetherin cytosolic lysines. 293T cells were co-transfected in duplicate with or without Vpu in the presence of either HA-tagged wild type tetherin or its counterpart having both its cytosolic lysines K18 and K21 replaced with arginines (KcytoR tetherin). Duplicate extracts were pooled and analyzed by western blotting. PCNA served as a loading control. Vpu-mediated tetherin depletion was quantified by densitometry, and a plot was generated from the results of two independent experiments performed in duplicate (right panel). The values obtained in the absence of Vpu were given the arbitrary value of 100%. Sizes of molecular weight markers are shown in kilodaltons.