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In vitro PARylation assays For the in vitro PARylation assay, purified GST-RSMC or GST-RSMC-16A was bound to Glutathione Sepharose 4B beads and incubated with the PARP1 enzyme (Sino Biological, 11040-H08B) for 1 h at 37 C in reaction buffer (50 mM TrisHCl pH 8.0, 150 mM NaCl, 10 mM MgCl 2 , 10% glycerol, 1 mM DTT) supplemented with or without 200 M NAD + , followed with three times wash with reaction buffer supplied with 300 mM NaCl

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In fact, at GSH concentrations within the physiological range in red blood cells, SNO-Hb can still promote vasodilation even under oxygenated conditions (46)