Fig 1.

Prediction of membrane topology and analysis of membrane-associated activity of DENV-2 NS2A. (A) Schematic representation of DENV-2 NS2A transmembrane segments predicted by HMMTOP, TMHMM2, SOSUI, DAS, TOPCONS, Split, and MEMSAT3. The gray boxes indicate predicted transmembrane segments (pTMS). The positions of the first and last amino acid of pTMS are indicated. (B) A reference model of DENV-2 NS2A topology. Different fragments covering the entire NS2A were C-terminally fused with eGFP. The amino acid positions of each NS2A fragment are indicated on the left. (C) IFA analysis of BHK-21 cells transfected with various NS2A-eGFP constructs. At 24 h p.t., the expression of eGFP was monitored by a mouse monoclonal antibody against eGFP and a goat anti-mouse IgG conjugated with Alexa Fluor 488. The eGFP signal is in white. (D) Membrane flotation analysis of 293T cells transfected with plasmids expressing NS2A fragment-eGFP fusion proteins. NS2A fragment-eGFP proteins in each fraction were detected using an antibody against eGFP. Calnexin, probed with rabbit IgG against calnexin (Sigma), was used as an integral membrane protein control. The percentages of signals detected in the low-density (LD) fractions (1 to 4) and high-density (HD) fractions (5 to 8) were calculated by ImageJ software and are indicated below the panels.