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. 2015 Apr 3;290(21):13234–13249. doi: 10.1074/jbc.M114.595462

FIGURE 1.

FIGURE 1.

Characterization of H2A.Z knockdown and the expression of a H2A.Z non-acetylatable mutant in C2C12 cells. A, Western blot analysis of whole-cell lysates from non-infected C2C12 cells or C2C12 cells stably expressing either a scrambled shRNA (shScramble) control or a shRNA targeting H2A.Z mRNA (shH2A.Z) using an anti-H2A.Z antibody. H2A.Z levels were reduced in the H2A.Z knockdown cells compared with cells expressing scrambled shRNA or normal cells. Histone H3 expression was used as a loading control. B, bright field microscopy images showing C2C12 H2A.Z knockdown cells after differentiation and incubation in differentiation medium for 96 h. Knockdown of H2A.Z did not affect the differentiation of C2C12 myoblast cells into myotubes. C, schematic showing H2A.Z-GFP and H2A.Z-Ac-mut-GFP, where the five acetylatable lysines in the N terminus were mutated to arginine. Blue letters represent known sites of acetylation at the N terminus of H2A.Z. Red letters represent sites of lysine to arginine mutations in H2A.Z-Ac-mut. D, Western blot analysis using anti-GFP antibody detects the stable expression levels of NLS-GFP, H2A.X-GFP, H2A.Z-GFP, and H2A.Z-Ac-mut-GFP in C2C12 cell lines. The numbers represent quantification of the GFP-tagged proteins on the Western blot using chemiluminescence imaging. Histone H3 expression was used as a loading control. E, Western blot analysis using an anti-H2A.Z antibody shows that H2A.Z-GFP and H2A.Z-Ac-mut-GFP were not overexpressed as compared with endogenous H2A.Z levels. The numbers represent quantification of GFP-tagged H2A.Z and H2A.Z-Ac-mut as well as endogenous H2A.Z on the Western blot using chemiluminescence imaging. H2A.Z-Ac-mut represents ∼8% of endogenous H2A.Z at the protein level. The asterisk on the Western blot shows a nonspecific band detected by the antibody. F, real-time PCR amplification of mRNA isolated from C2C12 stable cell lines. Relative GFP mRNA levels detecting expression of exogenous histones and NLS-GFP were normalized to HPRT. G, immunofluorescence images showing the nuclear staining of GFP-tagged H2A.Z, H2A.Z-Ac-mut, and H2A.X in the C2C12 stable cell lines. E, GFP ChIP showed that H2A.Z-GFP as well as H2A.Z-Ac-mut-GFP is enriched at the MyoD promoter as compared with an upstream region and the promoter of β-actin. Experiments were performed in high-serum (HS) growth conditions.