Journal of Signal Transduction / 2012 / Article / Fig 3

Research Article

Redox Regulation of Nonmuscle Myosin Heavy Chain during Integrin Engagement

Figure 3

Analysis of the redox state of muscle MHC and actin in differentiating myotubes. (a) Analysis of H2O2 content in myoblasts and in differentiating myotubes after six days of differentiation. Hydrogen peroxide assay was performed with DCF-DA. 𝑃 < 0 . 0 0 5 versus myoblasts. (b) Analysis of the redox state of MHC on rounded or spread human fibroblasts and in differentiating myotubes. BIAM labelled pattern of MHC was revealed by a Western blot using HRP-streptavidin conjugate after immunoprecipitation of nmMHC in human fibroblasts and mMHC in differentiating myotubes; MHC normalization was performed probing the membrane with specific anti-MHC antibodies; the histogram corresponds to the ratio between two corresponding values obtained by densitometric analysis. Ψ 𝑃 < 0 . 0 0 1 . (c) Analysis of MHC-actin association in myoblasts and in differentiating myotubes. An anti-nmMHC immunoprecipitation was performed from myoblasts and from differentiating myotubes. The amount of actin associated with MHC was obtained with antiactin immunoblot, while the normalization was performed using anti-MHC antibodies. The histogram reports the ratio between the values obtained by densitometric analysis of the two corresponding values of the blots. 𝑃 < 0 . 0 0 1 5 versus myotubes. (d) and (e) Analysis of actin glutathionylation. Actin glutathionylation was assayed on growing C2C12 myoblasts, differentiating myotubes, human fibroblasts, and in murine skeletal muscle, liver, and kidney using anti-GSH antibodies. Φ 𝑃 < 0 . 0 0 1 versus myotubes. a.u.: arbitrary units.

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