Fig. 2

The mutation (Arg76Pro) of MyoD has no effect on its transcriptional activity. a Schematic representation of WT pig MyoD protein. Arrows denote amino acid positions of the mutation. b qPCR analysis for the expression of myoD in NIH3T3 cells treated with overexpression Flag-MyoD^WT、Flag-MyoD^R76P or a control Flag vector. GAPDH was used as internal control. c Western blotting analysis for protein levels of MyoD by Flag antibody for 48 h in GM. d qPCR analysis was performed to detect the level of ckm、cdh15 and myh3 at 3d post-differentiation. e 293 T cells were transiently transfected with 4Rtk-luc and expression vector encoding the indicated proteins. Cells were harvested 48 h post-transfection. Firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System Kit (Promega). The data was presented as the normalized ratio of Firefly luciferase activity to the Renilla luciferase activity. f The myotube formation was observed under white light after differentiation for 5 days. Scale bar =50 μm. g After transfection and differentiation for 6 days, MyHC was detected by immunofluorescence staining. Scale bar = 100 μm. Data are presented as mean ± S.E.M.; n = 3; *p < 0.05, ***p < 0.001 (Student’s t-test)