Fig. 4

Stx4 and Cdo induce MyoD activities synergistically, and the Cdo-binding deficient Stx4 mutant failed to enhance myotube formation. a 10T1/2 cells were cotransfected with a MyoD-luciferase reporter and the expression vectors for MyoD and β-galactosidase as an internal control. In addition, control, Stx4, and/or Cdo expression vectors were cotransfected as indicated. Forty-eight hours later, the reporter activities were measured and normalized relative to the internal control. The experiment was performed as triplicates and repeated three times with similar results. *p < 0.01. b Lysates of C2C12 cells stably transfected with indicated Stx4 vectors were immunoblotted with antibodies to MHC and pan-Cadherin as a loading control. The relative signal intensities of MHC to pan-Cadherin were quantified and added under the blot. c C2C12 cells were transiently cotransfected with control (pcDNA), Stx4, or Stx4 mutants along with a GFP expression vector to mark the transfectant. Then, cells were induced to differentiate for 2 days, followed by immunostaining with an antibody to MHC and DAPI stain. Size bar = 100 μm. d Quantification of myotube formation of cell lines shown in panel c. Values represent means of triplicate determinations ± 1 SD. The experiment was repeated three times with similar results. Significant difference from control, *p < 0.01, **p < 0.005