FIG 4 .

The gp42 linker mutants bind exogenous gHgL and HLA class II comparable to wild-type gp42. CHO-K1 cells were transiently transfected with each of the d37-41 gp42 linker mutants. Twenty-four hours later, the cells were overlaid with sDQ2-αII (HLA class II) purified protein or sgHgL supernatants (isolated 48 h posttransfection). Protein was overlaid for 1 h at 4°C, and unbound protein was washed away. Binding of HLA class II and sgHgL was determined by cELISA using anti-HLA class II DQ antibody (1a3) (catalog no. ab24265; Abcam) and anti-FLAG-M2 (catalog no. F1804; Sigma), secondary biotinylated anti-mouse IgG antibody, tertiary streptavidin-HRP and TMB substrate and compared to expression using anti-gp42 antibody (3H3). Absorbance readings were taken at 380 nm using a PerkinElmer Victor plate reader. Binding was normalized to wild-type gp42 binding levels, which were set at 100%. Data shown are the average values of three independent experiments.