Fig. 5. Overexpression of RGMc or RGMcGPI does not alter muscle differentiation. (A) Immunohistochemistry for ectopic RGMc (anti-HA, green) in C2 myoblasts infected with Ad-tTA and either Ad-HA-RGMc or Ad-RGMcGPI, and incubated in differentiation medium for 48 hours (upper panels). The middle panels show an overlay of immunostaining with anti-HA and anti-myosin heavy chain (MHC; red) from the same microscopic field. Arrows indicate co-staining within multinucleated myotubes. The lower panels show results of immunocytochemistry for RGMc (anti-HA) and myosin heavy chain in C2 myoblasts infected with Ad-tTA and either Ad-HA-RGMc or Ad-RGMcGPI, as described above, and incubated in differentiation medium plus doxycycline for 48 hours (+ dox). Note that the extent of myotube formation and the levels of MHC are comparable whether or not wild-type RGMc or RGMcGPI are overexpressed. Magnification, 200x.