Fig. 2. Inhibition of differentiation markers and MyoD protein expression in Tax-expressing muscle cells. A, C2.7-Neo or C2.7-Tax cells were cultured in proliferation medium (PM) at 80% confluence, switched to differentiation medium (DM), and cultured for an additional 24, 48 or 72 h. At indicated times, cells were harvested, and cell extracts were prepared. Identical amounts of cell extracts (20 µg) were resolved by SDS-PAGE and then analyzed by immunoblotting as described in "Materials and Methods." The same membrane was probed sequentially with a polyclonal anti-Tax, anti-p21, antimyogenin antiserum, monoclonal anti-MHC, antidesmin, or an anti-GAPDH antibody, followed by the corresponding secondary HRP-coupled antibody. Antibody binding was visualized by enhanced chemiluminescence. B, C2.7-Neo (Lanes 1 and 2) or C2.7-Tax (Lanes 3 and 4) cells were cultured in proliferation medium (PM; Lanes 1 and 3) to 80% confluence, switched to differentiation medium (DM), and cultured for an additional 48 h (Lanes 2 and 4). The cells were then harvested, and total cell extracts were prepared. Protein separation and immunoblotting were carried out as indicated above. The same membrane was probed sequentially with polyclonal anti-Tax, polyclonal anti-Id1, polyclonal anti-Id2, and polyclonal anti-Id3 antisera, monoclonal anti-E2A, monoclonal anti-GAPDH, or monoclonal anti-MyoD antibodies, following by the corresponding secondary HRP-coupled antibody.