Fig. 6. The effect of protein X on the myogenic progression and Pax7 expression. (A) Primary myoblasts were incubated for 1 day in differentiation medium without protein X or with 2 µM protein X, in the absence or presence of 1 µM DAPT (D). Levels of Pax7, MyoD, myogenin, p21, and tubulin expression were examined by western blotting, band intensities were quantified by densitometry and are plotted on the right. Data represent mean values ± s.e.m. from three different experiments. (B) C2C12 cells were incubated for 1 day in differentiation medium without or with 2 µM protein X, and the levels of Pax7, MyoD, myogenin, p21 and tubulin expression were examined as in A. (C) Primary myoblasts incubated for 1 day in differentiation medium without or with 2 µM protein X were co-stained with rabbit anti-MyoD and mouse anti-Pax7 antibodies and with AMCA-conjugated anti-rabbit IgG and Rhodamine-Red-X-conjugated anti-mouse IgG antibodies. Two representative images are shown. The number of Pax7⁺/MyoD⁺, Pax7⁺/MyoD^– and Pax7^–/MyoD⁺ cells was counted in 15 different microscopic fields, 300-400 cells were analyzed for each experimental condition (without and with protein X). The data show mean values of cells counted on three different slides, error bars represent s.e.m. (*P<0.05). The experiment was repeated twice with similar results.