N-terminal sequencing of BST-2 ΔN20 NHA and glycosylation analysis.

(A) 293T cells were co-transfected with 500 ng of BST-2 ΔN20 IHA 159H6 or BST-2 ΔN20 NHA 159H6. After 48 h, cells were harvested and analyzed by Western blotting using an anti-BST-2 pAb, anti-His mAb and anti-tubulin mAb. (B) 293T cells in two T125 flasks were transfected with 6 µg of BST-2 ΔN20 NHA 159H6 per flask. Cells were harvested 48 h after transfection, lysed and separated by nickel affinity chromatography. Samples collected from the purification procedures were separated by SDS-PAGE with Coomassie Brilliant Blue staining and the concentrated BST-2 protein was labeled with an arrow. N-terminal sequencing results are shown below. (C) BST-2 WT or variant expression plasmids (500 ng) were transfected into 293T cells. Cells were harvested 48 h after transfection and divided into two portions. Cell lysates were incubated in the presence or absence of PNGase F and analyzed by Western blotting using an anti-BST-2 pAb and anti-tubulin mAb. This experiment was repeated for three times and the most representative data was shown.