FIGURE 3.
Deglycosylation of oligomannose-enriched SOSIP.681 gp140 trimers. A, SOSIP.681 proteins were produced in either kifunensine-treated 293T cells (upper panel) or 293S GnTI−/− cells (lower panel), treated with endoglycosidases Endo F1 or Endo H under native or denaturing conditions, and analyzed by SDS-PAGE. Deglycosylation reactions were performed in PBS at neutral pH (7.2) or in the buffer recommended by the manufacturer (sodium citrate, pH 5.5), as indicated. B, SDS-PAGE analysis of the gp41 ectodomain of mock- and Endo H-treated SOSIP.681GnTl−/−. The blots were probed with the anti-gp41 mAb D50 (left) or HIVIg (right). The positions of marker proteins are indicated. C, Endo H treatment of SOSIP.681GnTl−/− under mild conditions in the presence and absence of sCD4, 17b, or a combination of both ligands. D, BN-PAGE analysis of SOSIP.681 treated with or without Endo H under native conditions. The molecular masses of reference proteins are indicated.