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Figure 4


Fig. 4. Processing and localization of HA-tagged RGMc. (A) Detection of ectopic cell-surface RGMc by immunoblotting (arrows) in undifferentiated C2 myoblasts infected with either Ad-HA-RGMc (wt) or Ad-HA-RGMc{Delta}GPI ({Delta}) after labeling membrane proteins with the EZ-link reagent, followed by pull-down of protein extracts with streptavidin-agarose. The left panel shows immunoreactive RGMc in whole-cell protein extracts (WCE); results of pull-down experiments are displayed in the middle and right panels. Antibodies are as indicated. Immunostaining for cadherin is shown below. (B) Detection of RGMc released from the cell membrane following incubation of undifferentiated C2 cells infected with Ad-HA-RGMc with PI-PLC (+). Antibodies are as indicated. WCE is included for reference. For A and B, the gray arrow delineates the C-terminal fragment of RGMc. (C) Digestion of membrane-associated HA-RGMc with PI-PLC followed by PNGase F (+) or mock treatment (-). (D) Digestion of secreted HA-RGMc with PNGase F. Antibodies are as indicated. For C and D, white arrowheads indicate digestion products.





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