Fig. 2. Muscle-cell-associated RGMc is a GPI-anchored membrane protein. (A) Immunocytochemistry for cell-surface associated RGMc (red) in differentiated C2 myotubes (left panel). After pre-absorption of polyclonal anti-RGMc anti-serum with bacterially expressed RGMc bound to CNBr-Sephadex, no binding is seen (right panel). Nuclei in both panels are stained with Hoechst 33258 dye (blue). Magnification, 200x. (B) Detection of RGMc by immunoblotting (arrows) using membranes isolated from differentiated (T₇₂) but not undifferentiated C2 myoblasts (T₀), after separation by SDS-PAGE under reducing (upper left panel) and non-reducing conditions (right panel). Equivalent amounts of membrane proteins were used, based on equal detection of cadherin (lower left panel). (C) Detection of cell-surface RGMc by immunoblotting (arrows) after labeling membrane proteins of differentiating C2 myoblasts (72 hours in DM) with a cell-impermeable biotin crosslinking reagent (EZ-link), followed by pull-down of protein extracts with streptavidin-agarose (+, cells exposed to EZ link; -, mock-treated cells). Immunostaining for cadherin is shown below. (D) Detection of muscle RGMc (arrows) released from the surface of differentiated C2 cells (T72) after incubation with (+) or without (-) PI-PLC.