Fig. 8.

Effect of coculture of myotubes with NG108-15 cells on nPKC θ mRNA transcript expression. Primary myoblast cultures were established on 100 mm tissue culture dishes and cultured for 7 d as described in the legend to Figure 2. Myotubes were cultured in parallel for 2.5 d alone (PMC) or in the presence of 1.4 × 10⁶ NG108-15 cells (PMC + NG108-15). An equal number of NG108-15 cells was cultured separately in parallel (NG108-15). Total RNA from each culture and from adult skeletal muscle was isolated and quantitated. Total poly(A⁺)-enriched RNA from each culture was isolated, electrophoresed, and transferred to nylon membranes. Adult skeletal muscle poly(A⁺)-enriched RNA, obtained from an amount of total RNA equal to that present in the myotube culture (∼100 μg) (PMC), was loaded as a control (skeletal muscle). Blots were probed with³²P-labeled nPKC θ cDNA and exposed for autoradiography. Radioactivity was quantitated by PhosphorImager analysis using ImageQuant software. This figure represents one of three identical experiments in which the increase in nPKC θ mRNA in cocultured myotubes was 2.5-fold ± 0.44 SEM above control myotubes. The blot represented by the toppanel of this figure was subsequently reprobed with³²P-labeled α-actin cDNA. Arrowindicates skeletal muscle isoform of α-actin mRNA transcripts.