Necdin controls CCAR1 protein abundance in myogenic cells.

<p>A) CCAR1 mRNA relative quantification by qPCR using specific primers in C2C12 cells untransfected (Mock) or transfected with pCMV-FLAG-Necdin (Ndn) and in primary myoblasts from wt, Ndn<sup>−/−</sup> and tgMlcNec (MlcNec) mice (data representative of respectively four and three independent experiments). β-Actin was used as internal standards. B) Representative western blot showing CCAR1 and necdin (β-actin as loading control) expression in C2C12 mock transfected (M) or transfected with pSG5-HA-CCAR1 (C) and/or pCMV-FLAG-Necdin (N or N+C). Both the endogenous and transfected CCAR1 were detected with the polyclonal anti-CCAR1, the transfected CCAR1 with anti-HA. C) Graphs show mean values ± s.e.m. obtained from the ratio of densitometric values of protein/β-actin bands on the blots in (B). Data are representative of three independent experiments. (* <i>p</i><0,001 vs mock transfected; + <i>p</i><0,005 vs CCAR1 transfected). D) Representative western blot showing expression of CCAR1 and necdin in primary myoblasts isolated from wt, Ndn<sup>−/−</sup>, tgMlcNec newborn mice. Proteins were detected using antibodies specific for CCAR1, necdin and β-actin as control. E) Graphs show mean values ± s.e.m. obtained from the ratio of densitometric values of protein/β-actin bands on the blot of the same experiments. Data are representative of three independent experiments. (* <i>p</i><0,002 vs wt). F) Co-localization of necdin and CCAR1. Images taken at confocal laser scanning miscoscope (63x magnification, zoomed 1.67 times) showing co-immunostaining on C2C12 transfected with pSG5-HA-CCAR1 (i) and/or pCMV-Ndn (C2C12, CCAR1-Ndn-N+C) (ii, iii) and on primary myoblasts from Ndn<sup>−/−</sup> (Ndn<sup>−/−</sup>) (iv) and tgMlcNec (MlcNec) (v) newborn mice. Panels i-ii-iii-iv-v show immunostaining using the specific monoclonal anti-Ndn (Ndn: Ab-Ndn-green); panels i'-ii'-iii'-iv'-v' show immunostaining using the polyclonal anti-CCAR1 (CCAR1: Ab-CCAR1-red). Co-immunostained images of anti-Ndn and anti-CCAR1 are shown in panels i''-ii''-iii''-iv''-v'' (Ab-CCAR1 + Ab-Ndn-yellow). Panels i'''-ii'''-iii'''-iv'''-v''' show merged images with nuclei stained with DAPI (merge Ab-CCAR1 + ab-Ndn + DAPI). Scale bars (i–iii) 41,75 µm; (iv–v) 44,7 µm.</p>