(A) 293T/tetherin or 293T/tetherin Y6,8A mutant were infected with VSV-G-pseudotyped HIV-1 wt, HIV-1 delVpu or HIV Vpu ELV at an MOI of 0.5. Supernatants and cell lysates were harvested 48 h later and analyzed for infectious virus release on HeLa-TZMbl as in Figure 1C. (B) Corresponding Western blots of cell lysates and virions from A. (C) 293T/tetherin Y6,8A cells treated with control or UBAP1 specific siRNAs were infected with VSV-G-pseudotyped HIV-1 wt, HIV Vpu ELV, HIV-1 Vpu A14L/W22A or HIV-1 delVpu at an MOI of 2. 48 h post infection, cells were lysed and immunoprecipitated with an anti-tetherin antibody. Lysates and immunoprecipitates were subjected to SDS-PAGE and analyzed by Western Blotting for tetherin, UBAP1 and Vpu, and analyzed by LiCor quantitative imager. Ratios of Vpu/tetherin band intensities in the coIP are plotted on the histogram below.