Figure 3

Purification of sGP1-FLAG, GPCΔTMΔIC-FLAG, and GPCΔTM-FLAG from transiently transfected HEK-293T/17 cell supernatants. Supernatants from 72-hour transient transfections were subjected to IP using an ANTI-FLAG M2 agarose gel. Twenty microliters of cleared supernatant (lane 1), unbound fraction (lane 2), and eluate fraction (lanes 3) were resolved on 10% SDS-PAGE gels, blotted onto nitrocellulose membranes and probed with anti-FLAG M2 mAb and a goat α-mouse IgG-HRP (panel A). The blot was stripped and reprobed with anti-GP1 mAb 2074 and the same secondary as above (panel B). The monomer sGP1 and sGP2 species (m) on each blot are indicated by solid arrows. The GP1+GP2 polyprotein on each blot (poly) are indicated by broken arrows. MagicMark XP western blot molecular weight markers (M), with sizes (kDa) are shown to the left of the panel. The construct designations for each transfection supernatant are indicated below the figure. In both panels lanes 1, 4, and 7 correspond to supernatant load; lanes 2, 5, and 8 are unbound supernatant fractions; lanes 3, 6, and 9 are eluate fractions.