Figure 5
Cross-linking Nef dimers in intact cells with formaldehyde. (A) 293T cells were transfected to express SF2-HFNef or SIVMAC239-HFNef. Intact cells were incubated with 0.5% formaldehyde for 10 minutes at room temperature. Clarified whole cell lysates were prepared and Nef detected by Western blot analysis. (-), incubation without formaldehyde. (+), incubation with formaldehyde. Arrows, cross-linked Nef dimers. (B) Assay of intracellular oligomerization with NA7Nef/SF2Nef chimeras. The chimeric expression vectors were made by substituting coding sequence for amino acids 24-200 SF2Nef into NA7Nef and the same coding sequence for NA7Nef into SF2Nef. Oligomerization was assayed by formaldehyde cross-linking in intact cells as in (A). NA7(SF2)Nef, NA7Nef with coding sequence for amino acids 24-200 replaced by coding sequence for the same amino acids from SF2Nef. SF2(NA7)Nef, the reciprocal construct as NA7(SF2)Nef. (-), incubation without formaldehyde; (+), incubation with formaldehyde. Arrow, cross-linked Nef dimer. (C), The ability of SF2NefG2A to associate with itself was compared to SF2Nef by the formaldehyde cross-linking assay with intact cells. Arrow to the right of lane 4 indicates cross-linked Nef (lanes 2 and 4). (+), Cells incubated with 0.5% formaldehyde for 10 minutes at room temperature. (-), Cells incubated in the absence of formaldehyde.