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Fig. 6. FHL3 forms a complex with MyoD and other bHLH proteins. (A) C2C12 cells were co-transfected with various combinations of expression vectors for Flag-vector, MyoD-Flag, Gal4DBD and/or Gal4DBD-FHL3, together with Gal4-luciferase and Renilla luciferase reporters. Transfected myoblasts were maintained in growth media for 24 hours then differentiated for 48 hours and assayed for luciferase activity. Luciferase values were corrected for background luminescence, normalised to Renilla luciferase activity to adjust for transfection efficiency and standardised relative to Gal4DBD expressing samples (arbitrarily defined as 1 relative luciferase unit RLU). Error bars represent ± s.e.m. (n=4, ***P<0.001). White boxes represent GAL4DBD-FHL3 and black-line just above baseline represents GAL4DBD. (B) Lysates from C2C12 myoblasts transfected with pCMX-Gal4DBD or pCMX-Gal4DBD-FHL3 for 24 hours were immunoblotted to confirm recombinant protein expression. (C) Lysates from C2C12 myoblasts transfected with pEFBOS-Flag or pEFBOS-MyoD-Flag for 24 hours were immunoblotted to confirm recombinant protein expression. (D) GST-FHL3 and His-MyoD were coexpressed in E. coli. GST-proteins and associated proteins were bound to glutathione Sepharose, washed extensively and eluted with reduced glutathione. Eluted proteins (bound) and whole-cell extracts (input fractions) were immunoblotted for recombinant GST, or His protein expression. Western blots are representative of three experiments. (E) GST or GST-FHL3 captured on glutathione-Sepharose was incubated for 4 hours, with cell lysates from C2C12 cells differentiated (24 hours), washed extensively and immunoblotted for endogenous MyoD, or recombinant GST and/or GST-FHL3 using antibodies as indicated. Results shown are representative of three similar experiments. S/N and input fractions represent unbound lysate and C2C12 cell lysates prior to incubation with GST or GST-FHL3 respectively. (F) GST or GST-FHL3 pull-downs described above in (E) were also immunoblotted for endogenous Myf5, myogenin, and E47 using indicated antibodies. Western blots are representative of three experiments. (G) C2C12 myoblasts were stained with anti-FHL3 (green), anti-MyoD antibodies (red), and with the nuclear stain To-Pro-3 (blue) and imaged by confocal microscopy. Bar, 20 µm.
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