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. Author manuscript; available in PMC: 2016 Oct 1.
Published in final edited form as: Biochim Biophys Acta. 2015 Aug 18;1849(10):1277–1288. doi: 10.1016/j.bbagrm.2015.08.004

Figure 6. Arsenite induces JNK-dependent H3S10 phosphorylation and recruits Nrf2 to the HO-1 ARE prior to enrichment of phosphorylated H3S10.

Figure 6

Figure 6

a) HaCaT cells were transiently transfected with JNK specific siRNA or control siRNA as described in Materials and Methods. After 48 hr, cells were treated with 10µM arsenite for 8 hr and nuclear and cytoplasmic fractions were subjected to Western blot analysis with anti-HO-1, anti-Nrf2, anti-JNK, and anti-H3S10 phospho-specific (H3S10-P) antibodies; Lamin B and β-Actin blots are shown as loading controls. Quantification of blots is shown to the right. Densitometry analysis was conducted with Image J software. Relative density of arsenite treated signal bands were normalized to the corresponding control signal bands (siCon/arsenite (−)) for fold change, then further normalized to the relative density of loading control signal bands for loading accuracy; JNK and HO-1 were normalized to β-Actin; H3S10-P and Nrf2 were normalized to Lamin B. (b and c) HaCaT cells were treated with 10µM arsenite for 0.5, 1, 2, or 4 hr, then harvested for ChIP assay and incubated with rabbit IgG, anti-Nrf2 (b), or anti-H3S10 phospho-specific (H3S10-P) (c) antibody. Isolated genomic DNA was subjected to quantitative RT-PCR using primer pairs for the HO-1 E1 region. Samples were normalized to input and presented as percent input. Significance was calculated using a t-test and established with p < 0.05. A representative of three independent experiments is shown. (d) Graphical representation of temporal induction of Nrf2 and phosphorylated H3S10 (H3S10-P) enrichment on the HO-1 E1 ARE.