Figure 3.

Effects of GSSG-dependent thiol-to-disulfide exchange on fusion and gp64 trimerization. (A) GSSG after DTT treatment, in absence of alkylation, fully recovered fusion. Op1D cells were first treated with 10 mM DTT for 30 min followed by a 15-min application of 1–30 mM GSSG. PKH26-labeled RBC were then added to Op1D cells and the medium was acidified (i.e., pH 5.1) to trigger the fusion. Each bar represents mean fusion extent ± SE for 200 cells counted. (B) Western blot analysis GSSG-treated Op1D cells. Cells were lysed in nonreducing 1.5% SDS lysis buffer and proteins were separated by 4–12% gradient SDS-PAGE. GSSG, after DTT, treatment restored gp64 trimers. (C) Quantitative Western blot analysis of GSSG-oxidized gp64 immunoreactive bands. Relative band intensities, measured by ECF, are presented as a percentage of the total band intensity within the sample lane. Restoration of trimer 1 was greater than that of trimer 2.