Figure 6
Cross-linking Nefs dimers with BM[PEG] 3 . (A) 293T cells were transfected to express SF2Nef, SF2NefC55S and SF2NefC206X (left, lanes 1-10), and SIVMAC239Nef (right, lanes 11 and 12). Clarified whole cell Iysates were incubated with BM[PEG]3. Nef was then detected by Western blot analysis. (-), clarified whole cell Iysates incubated in the absence of BM[PEG]3 for 1 hour at room temperature (lanes 1-5 and 11). (+), clarified whole cell Iysates incubated in the presence of 0.5 mM BM[PEG]3 for 1 hour at room temperature (lanes 6-10 and 12). The presence of crosslinked Nefs was detected by Western Blot analysis. NT, 293T cells not transfected. (B) SF2Nef, SF2C55S, and SF2C206X were assayed for CD4 downregulation and MHCI downregulation in transduced CEM cells (top). The level of expression of SF2Nef, SF2NefC55S and SF2NefC206X in CEM cells was determined by Western blot analysis (bottom). (C) Lysates from transfected 293T cells expressing SF2NefC206X, SF2NefC55S, and SF2Nef were cross-linked by 0.5% formaldehyde.