Fig. 5: Canonical biosynthesis of gB was disrupted by V528P.

a MeWo cells transiently expressing WT gB and gB mutants were imaged by confocal microscopy at 24 hrs post transfection. Cells were stained with mouse anti-gB mAb SG2 (red), trans-Golgi network marker, TGN46 (green), or early endosome antigen, EEA1 (green), and nuclei were stained with Hoechst 33342 (blue). Representative images are from experiments performed at least in duplicate. Areas in white boxes are shown at higher magnification in panels to the right; white arrows indicate gB colocalization with TGN46 or EEA1 respectively. Representative examples from 5 fields of view recorded from n = 3 independent experiments for proline mutants and n = 2 independent experiments for alanine mutants are shown. Scale bar = 15 µm. b Intracellular trafficking of gB was assessed by glycosylation status. MeWo cells transfected with vectors expressing WT gB, or gB mutants were lysed at 24 hrs post transfection and immunoprecipitated with anti-gB mAb SG2, left untreated (-), or treated with Endo H (H) or PNGase F (F). Proteins were separated by SDS-PAGE gel (7.5%) under reducing conditions, and gB was detected with rabbit anti-gB 746-867. The arrows and red vertical lines indicate the glycosylated forms of full-length and cleaved gB respectively, the blue vertical lines indicate the Endo H-resistant glycoforms of cleaved gB, and arrowheads indicate forms of gB deglycosylated by both Endo H and PNGase F. Representative examples from n = 2 independent experiments are shown. Mass markers (kDa) are shown on the left. Source data are provided as a Source Data file.