Fig. 2.

Subcellular fractionation experiment of the MH domain mutants of DENV4 prM/E construct. (A) 293T cells transfected with mock, WT or mutants were washed with PBS, resuspended in modified buffer B, and frozen-thawed 8 times (Hsieh et al., 2008; Xu et al., 1997). After clearing the nuclei and debris, the membrane fraction, pellets derived from the soluble fraction by ultracentrifugation and pellets derived from culture supernatants were subjected to Western blot analysis using human serum of a dengue case (upper gels) (Wang et al., 2006), followed by reprobing with anti-calnexin Mab (lower gels). The long exposure gels for prM bands in pellets were shown (middle gels). (B) Membrane fraction and pellets of the soluble fraction of WT and mutants were treated with endo H (H) or PNGase F (F), and subjected to Western blot analysis as in panel A. Arrow heads indicate E or deglycosylated E protein (Edg). The size of molecular weight markers is shown in kDa. One representative experiment of three is shown.