Fig. 6. Biosynthesis and processing of RGMc and RGMcGPI in Cos-7 cells. (A) Time course of accumulation of RGMc in whole-cell protein extracts (WCE) and conditioned culture medium (CM) after infection with Ad-HA-RGMc. Full-length and internally cleaved RGMc proteins are indicated by black or gray arrows, respectively. Immunoblots for tubulin and albumin protein staining (lower panels) indicate equivalent sample loading. (B) RGMc rapidly accumulates at the cell membrane following its biosynthesis. RGMc was analyzed by immunoblotting at the indicated times following infection with Ad-HA-RGMc after labeling of cell membranes with the EZ-link reagent and streptavidin-agarose pull-down. Immunostaining for tubulin and cadherin (lower panels) is included for loading and cell-surface specificity controls, respectively. (C) Time course of accumulation of RGMc in WCE and CM after infection with Ad-HA-RGMcGPI. Full-length and internally cleaved RGMc proteins are indicated by black or gray arrows, respectively. Immunoblots for tubulin and albumin protein staining (lower panels) are included to demonstrate equivalent sample loading. (D) Immunostaining with anti-HA antibody of CM samples from C.