HDAC3 activity

HDAC3 activity was measured by measuring HDAC activity on DRGs after central and peripheral injury prior to HDAC3 immunoprecipitation. Briefly, sciatic DRGs from 3 animals were extracted 24 h after surgery and pooled per sample, protein lysates were obtained using non‐denaturizing lysis buffer (NDLB, 20 mM Tris–HCl pH 8, 137 mM NaCl, 1% IGEPAL, 2 mM EDTA), and for the HDAC3 immunoprecipitation, total lysates were bound to HDAC3 antibody (rabbit, Abcam), pulled down with Protein G Magnetic Beads, washed with NDLB, eluted with 0.1 M glycine pH 2 and neutralized with Tris pH 8. Activity of samples of 20 μg of immunoprecipitated HDAC3 was measured with fluorometric histone deacetylase activity kit (Active Motif, Rixensart, Belgium) according to the manufacturer's guidelines.