Figure 6

PNGase F and Endo H treatment of sGP1-FLAG, GPCΔTMΔIC-FLAG, and GPCΔTM-FLAG from transfected cell supernatants. Twenty μL of each FLAG M2 resin-purified protein from a typical transfection, as shown in figure 3, were subjected to treatment with 500 U of PNGase F or Endo H, as described in materials and methods. The reactions were resolved on SDS-PAGE gels alongside untreated counterparts, blotted, and probed with anti-FLAG M2 mAb and an HRP-labeled goat α-mouse IgG. Soluble GP1-FLAG (lanes 1, 2, 3), GPCΔTMΔIC-FLAG (lanes 4, 5, 6), and GPCΔTM-FLAG (lanes 7, 8, 9) show different mobilities based on whether proteins were treated with PNGase F (lanes 2, 5, 8), Endo H (lanes 3, 6, 9), or were left untreated (lanes 1, 4, 7). MagicMark XP™ protein molecular weight markers (M), with sizes (kDa) are shown to the left of the panel. Glycosylated species are denoted by (+) CHO, and deglycosylated counterparts by (-) CHO. The addition of amydase to each reaction is denoted by (+) symbol below the figure, whereas untreated controls are marked by (-).