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Fig. 8. Treatment of histone H4 with peptidylarginine deiminase results in loss of staining for the arginine 3 histone H4 methyl modification. Histone H4 was purified from 293T cells and treated with either heat-inactivated or active skeletal muscle peptidylarginine deiminase (smPAD) and levels of methylated arginine 3 on histone H4 [ ${alpha}$ Me(Arg3)H4], hyperacetylated histone H4 ( ${alpha}$ Hyperacetyl H4), and citrulline ( ${alpha}$ -Mod-Cit) were then detected by western blot analysis. The blots were stained with Ponceau S prior to western analysis and the smPAD and histone H4 (as well as a lower amount of H2A that co-purified with H4) could be visualized. The mobility for H4 and H2A increased following PAD treatment and these modified forms are indicated by asterisks on the Ponceau S stained blot. Staining for the histone Me(Arg3)H4 modification was observed following treatment with heat inactivated smPAD whereas no staining was observed for this modification following treatment with active smPAD. Staining for the hyperacetylated H4 modification appeared to be only slightly reduced (if at all) following smPAD treatment. Citrullination of histone H4 increased dramatically following smPAD treatment. Other contaminating proteins in the H4 sample, including what is probably H2A, were also citrullinated. The citrullinated protein in the heat-inactivated smPAD (control sample) probably indicates that smPAD is capable of deiminating itself. These results provide indirect evidence that the observed loss of staining for methylated arginine residues on egg and embryonic histones was a result of PAD activity.

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