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Necdin controls CCAR1 protein abundance in myogenic cells.

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posted on 2012-08-14, 00:44 authored by Stephanie François, Cristina D'Orlando, Tiziana Fatone, Thierry Touvier, Patrizia Pessina, Raffaella Meneveri, Silvia Brunelli

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−/− 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. (* p<0,001 vs mock transfected; + p<0,005 vs CCAR1 transfected). D) Representative western blot showing expression of CCAR1 and necdin in primary myoblasts isolated from wt, Ndn−/−, 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. (* p<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−/− (Ndn−/−) (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.

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