Expression of E1 mutants. (A) Western blotting analysis. 293T cells were transfected with pcDNA3, marked as the control, or with each of the pcDNA3-dervied WT and mutant E1E2 plasmids. Cell lysates containing equal total proteins prepared 2 days after transfection were subjected to reducing SDS-PAGE followed by Western blotting using E2- and E1-specific MAbs, respectively. (B to D) Analyses of mutant protein expression by FACS. 293T cells in two sets were transfected with pcDNA3 (marked as the control) or with each of the WT and mutant proteins. Two days after transfection, one set of cells was assessed for total E1 expression (B) and the other for E1 cell surface expression (C) using an E1 MAb. In another separate experiment, transfected cells were assessed for the total and cell surface expressions of E2 with a rabbit anti-E2 antibody (D). (E) Analyses of Cys-substituted mutant proteins by reducing and nonreducing SDS-PAGE. 293T cells transfected with pcDNA3 (marked as the control), WT, C272A, and C281A mutant plasmids were resolved by reducing or nonreducing conditions as indicated. The Western blots were then analyzed with E1- and E2-specific MAbs, respectively. The sizes in kDaltons of standard proteins were indicated to the right.