a, E2-specific western blot of cell culture supernatants showing secreted protein levels of eE2 mutants I422A, Y529A, W531A, and double mutant Y529A/W531A. Expi293 GnTI− cells were transfected and supernatants (uncleaved eE2 protein) were mixed with reduced 2x sample buffer. 15 ul of supernatant was loaded in each well. E2 2C1 primary antibody was used for western blotting. b, Coomassie-stained 4-20% Bis-Tris SDS-PAGE gels of purified eE2 mutant proteins in the presence (Reduced) and absence (Non-reduced) of β-mercaptoethanol. c, E2-specific western blot of top fractions from liposome flotation assays, comparing increased loading (as labelled under each blot) of mutants. Protein molecular weight maker (L) and wild-type eE2 is provided as a marker (std). Sample pH, inclusion of tCD81-LEL, and eE2 mutant proteins are labelled.