Fig. 6.
Effect of myotube indirect coculture with NG108-15 cells on PKC θ and α expression. Primary myoblast cultures were established and cultured for 7–8 d as described in the legend to Figure 2. Myotubes were cultured in parallel for 2.5 d alone (PMC) or were indirectly cocultured with 5 × 105 NG108-15 cells (NG108-15) separated from the myotubes (PMC*) by a semipermeable membrane of 3 or 8 μm pore size as indicated. After 3 d, myotube cultures and NG108-15 and myotube indirect cocultures were each harvested separately and homogenized, and protein (120 μg for nPKC θ, 50 μg for cPKC α) from cytosolic and membrane fractions from each cell population was analyzed as described in the legend to Figure1. Immunoblots were probed with anti-nPKC θ antiserum (A,B) or anti-cPKC α antibody (C, D) as described in the legend to Figure 2. Each lane represents a separate 35 mm tissue culture dish (PMC) or cell populations present in separate compartments of individual coculture experiments performed in triplicate (NG108-15, PMC*).