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. 2015 Apr 15;89(13):6746–6760. doi: 10.1128/JVI.00423-15

FIG 2.

FIG 2

DENV NS4B is an N-glycosylated protein in virus-infected cells. (A and B) NS4B is modified by N-glycosylation in DENV-infected cells. BHK-21 cells (4 × 105 cells in 6-well culture dish) were infected with DENV2 (IF) or heat-inactivated virus (H.I.) at an MOI of 10 for 48 h. For panel A, 50 μg of total cellular proteins was resolved by 12% SDS-PAGE. For panel B, 50 μg of total cellular proteins from virus-infected cells (IF) was digested with PNGase F or not and resolved by 12% SDS-PAGE. (C to F) Enrichment of glycoproteins by ConA agarose and PNGase F treatment of putative NS4B glycosylated form. BHK-21 cells for panels C, D, and E and Huh7 cells for panel F (8 × 106 cells in 10-cm cell culture dish) were infected (IF) with DENV2 or heat-inactivated virus (H.I.) at an MOI of 10 for 48 h. Cell lysates were harvested, and 3 mg of total cellular proteins was applied to ConA agarose for glycoprotein enrichment according to the procedures described in Materials and Methods. For panel C, 2% of the starting material (input), 2% of the unbound fraction (unbound), and 80% of the E1 elution fraction were resolved by 10% SDS-PAGE for Western detection of NS4B protein. For panel D, 0.35% of the starting material (input), 0.35% of the unbound fraction (unbound), and 5% of the elution fractions E1 and E2 were resolved by nonreducing 10% SDS-PAGE for Western detection of envelope (E) protein. For panels E and F, 30% of the E1 elution fraction from IF or H.I. cell lysates was digested with PNGase F or not and resolved by 12% SDS-PAGE. Lanes 1 of panels B, E, and F contain the PNGase F enzyme only. DENV NS4B protein was detected by polyclonal anti-rabbit NS4B antibody, DENV E protein was detected by monoclonal anti-E (4G2) antibody, and GAPDH served as a loading control. “<” indicates the signals derived from NS4B protein, “≪” indicates the signal derived from the putative glycosylated form of NS4B protein, and “<-” indicates the signal derived from PNGase F. All experiments were performed three times, and the data shown are from one representative infection experiment.