Fig. 2

MSY3 is phosphorylated by Akt in C2C12. a Left: multi-band migration pattern is shown in the Western blot with protein extracts of C2C12 myoblasts (GM) and myotubes (DM) treated with 1 unit of Antarctic Phosphatase (AP), probed with anti-MSY3 Ab (ZONAB). α-tubulin was used as normalizer. Right: densitometry calculations for faster (dephosph) and slower (phosph) migration bands of the Western blot on the left, normalized by α-tubulin. b Left: Western blot probed with anti-MSY3 Ab (ZONAB) and anti-myogenin Ab with protein extracts of C2C12 myoblasts (GM) treated with two different (20 μM and 30 μM) concentrations of LY294002 (LY). α-tubulin was used as normalizer. Right: densitometry calculations for faster (dephosph) and slower (phosph) migration bands of the Western blot on the left, normalized by α-tubulin. c Left: immunoblot with an anti-Akt total, an anti-MSY3 and anti-myogenin Abs in protein extracts of C2C12 grown in DM for 36 h, transfected with scrambled RNAi oligos (Ki) and RNA interfering oligos against of Akt 1 and Akt 2 (Akti), demonstrates that Akt activity is responsible for MSY3 phosphorylation. Right: densitometry calculations for Akt, myogenin, and MSY3 faster (dephosph) and slower (phosph) migration bands of the Western blot on the left, normalized by α-tubulin. We estimated a reduction of Akt of 75 %. Figure displays results representative from three independent RNAi experiments. Akt P <0.01; MSY3 dephosph P <0.05; MSY3 phosph P <0.01; myogenin P <0.05 by Student’s t test