Role of miR-200c in Myogenic Differentiation Impairment via p66Shc: Implication in Skeletal Muscle Regeneration of Dystrophic mdx Mice

Figure 4

miR-200c overexpression induces p66Shc phosphorylation in Ser-36. C2C12 myoblasts were infected either with a lentivirus encoding miR-200c or with a control virus. After selection with puromycin, cells were transfected with 1 μg of p66wt or a mutated version in which Ser-36 was substituted with Ala that was no longer phosphorylable (p66mut). (a) A representative Western blot using p66Shc-Ser-36 antibody showed that p66wt phosphorylation was higher in miR-200c-overexpressing cells compared to scramble control both without and with H₂O₂ treatment. Phosphorylation of p66mut was not present, as expected, in any condition. α-Tubulin (TUB) was used as loading control. (b) Bar graph showing the quantification of p66Shc phosphorylation in Ser-36 versus p66wt protein levels of C2C12-overexpressing miR-200c compared with control cells (; ). (c) C2C12 myoblasts were infected either with a lentivirus encoding miR-200c or with a control virus. After selection with puromycin, cells were immunoprecipitated (Ip) with either an anti-p66 antibody or an irrelevant isotypic antibody (negative control). Western blotting with a p66Shc-phospho-Ser-36 antibody revealed that p66Shc was more phosphorylated in Ser-36 in miR-200c IP-p66 than in scramble control cells. The efficiency of immunoprecipitation was assessed with an anti-p66 antibody. One-twentieth of the immunoprecipitated whole-cell extract (input) was loaded as a reference. (d) Bar graph showing the quantification of p66Shc phosphorylation in Ser-36 versus p66 total protein levels of C2C12-overexpressing miR-200c compared with scramble control cells (; ).

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