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. 2021 Sep 1;22(17):9509. doi: 10.3390/ijms22179509

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© 2021 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

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Neddylation is differentially regulated during C2C12 differentiation. (A–C) Western blot of the indicated proteins (A), and quantifications of NAE1, UBC12, and neddylated Cullins (N8-CULs) (B); quantifications of MYOD1, MYOGENIN, and MYOSIN (skeletal, fast) (MyHC) (C) in proliferative C2C12 cells cultured in growth media (i.e., day 0, D0) and for the indicated days after differentiation induction (D1 to D5). Proteins were quantified and normalized to ACTB. Data were presented as fold changes with D0 cells set at 1. N8-Conj.: NEDD8-conjugated. Arrows indicate neddylated CULs and CUL1. (D) Representative immunofluorescence images of C2C12 cells stained with NEDD8 (green) in growth media (D0) and for 3 and 6 days in differentiation media (D3, D6). Nuclei were counterstained with Hoechst 33342. Scale bars: 50 μm. ** p < 0.005 versus D0. One-way ANOVA. Three independent experiments.