FIG. 2.

PrM/E expression constructs, surface expression, and glycosylation pattern. (A) DENV2 PrM/E expression construct (pCB-D2) and chimeric construct containing the TMD and CY domain of CD4 in the TMD (D2CD4). ss, signal sequence. (B) 293T cells transfected with each of these constructs were permeabilized with or without Triton X-100 and subjected to flow cytometry analysis using anti-E MAb FL0232. Solid lines indicate anti-E MAb without Triton X-100, and solid lines with a shaded area indicate anti-E MAb with Triton X-100. Dotted lines indicate isotype controls, which overlap with those of mock-transfected cells (not shown). The relative surface expression of E protein was the ratio of the percentage of FL0232-positive cells in the absence of Triton X-100 to that in the presence of Triton X-100. (C) Lysates derived from 293T cells transfected with the constructs described for panel A were treated with endo H (H) or PNGase F (F) and subjected to Western blot analysis by using serum from a confirmed DENV2 case (70). Arrowheads indicate E or deglycosylated E protein (Edg). Molecular mass markers are shown in kDa. One representative experiment of three is shown.