Fig. 3. Increased phosphorylation of MyoD in Tax-expressing cells does not correlate with increased MyoD degradation. A, C2.7 (Lanes 1 and 4), C2.7-Neo (Lanes 2 and 5), and C2.7-Tax (Lanes 3 and 6) cells were cultured in proliferation medium (PM; Lanes 13) or differentiation medium (DM; Lanes 46) for 2 days. cdk-2 was immunoprecipitated from cell extracts by an anti-cdk2 antibody. The cdk2 kinase activity was tested against H1 histone as a substrate, using [-32P]ATP. The kinase reaction mixture was then resolved by 15% SDS-PAGE and transferred to a PVDF membrane. Phosphorylated radioactive H1 histone was visualized after autoradiography. The same membrane was exposed either overnight (H1) or for 48 h (H1*). The presence of immunoprecipitated cdk2 was confirmed by immunoblotting of the same membrane (WB cdk2). In control reactions (Lanes 712), the anti-cdk2 antibody was omitted during the immunoprecipitation step. The amount of radioactive phosphorylated H1 histone was quantified by phosphorimager. The result of this quantification is shown in the graph at the bottom. B, C2.7-Neo (Lanes 15 and Lane 11) and C2.7-Tax (Lanes 610) cells were cultured in proliferation medium (PM) without or with 15 µg/ml cycloheximide for the indicated times and then processed for Western blot analysis. Cell extracts (100 µg of total proteins) were resolved by SDS-PAGE and transferred to a PVDF membrane, as described above. This membrane was probed sequentially with a polyclonal anti-Tax antiserum, a monoclonal anti-MyoD antibody (5.8A), or a monoclonal antidesmin antibody (DE-U-10), followed by the corresponding secondary HRP-coupled antibody. Antibody binding was visualized by enhanced chemiluminescence. The intensity of the MyoD protein relative to that of desmin was plotted against the time of culture in cycloheximide (bottom). The half-life was determined as indicated in "Materials and Methods." Western blots were scanned, and the band intensities were quantified by the Image-Quant software. C, C3H10T1/2 cells were cotransfected with the MyoD and Tax expression plasmids (Lanes 3 and 4) or cotransfected with the MyoD expression vector alone (Lanes 1 and 2). Twenty-four h after transfection, the cells were further cultured in proliferation medium (PM; Lanes 1 and 3) or in differentiation medium for 2 days (DM; Lanes 2 and 4) and then harvested and lysed in antiphosphatase-containing buffer (see "Materials and Methods"). Twenty µg of total cell lysates were used in a Western blot analysis with a monoclonal anti-MyoD antibody. The slower migrating form of MyoD corresponds to the MyoD protein phosphorylated on Ser200 (13)
. D, C3H10T1/2 cells were cotransfected with the MyoD and Tax expression plasmids (Lanes 610) or transfected with the MyoD expression vector alone (Lanes 15) and further cultured in growth medium for 2 days. Cells were then cultured in the presence of cycloheximide and analyzed as described for B. Twenty µg of total cell lysates were used for Western blot analysis with monoclonal anti-MyoD or antiactin antibodies or a polyclonal anti-Tax antiserum. The intensity of the MyoD protein relative to that of actin was plotted against the time of culture in cycloheximide (bottom). The half-life was determined as indicated in "Materials and Methods." Blots were scanned, and the band intensities were quantified by the Image-Quant software. The data presented are from one typical experiment of four performed.