Western blot analysis reveals that Krüppel-like factor 6 (KLF6) and Myocyte enhancer factor 2 (MEF2D) are co-expressed in C2C12 myoblasts. (a) Myoblasts were cultured in growth medium (10% serum), followed by serum withdrawal (2%) for 144 h and harvested at 24-h time intervals. Cells were then lysed and equal amounts of protein (20 μg) were used for western blot analysis. The levels of the indicated proteins were assessed by a standard immunoblotting technique using specific primary antibodies for each. Actin was used as a loading control. (b) Immunocytochemistry reveals that KLF6 and MEF2D are co-localized in the nucleus at the myoblast stage but to a lesser extent in differentiated myotubes. C2C12 cells were treated as previously described by Salma and McDermott, 2012 . We used 4’,6-diaminidino-2-phenylindole (DAPI) staining for nuclear staining; green and red were used for MEF2D and KLF6 respectively and were then merged. (c) Transforming growth factor β (TGFβ) treatment potentiates the KLF6 promoter region through MEF2. KLF6 promoter constructs (pROM6 Luc and pROM6 ΔMEF2 Luc) were used, and Luciferase activities were analyzed upon serum withdrawal, with and without 2 ng/ml TGFβ treatment as indicated.