Nurr1

Frozen midbrain samples were crushed to a fine powder and crosslinked in fixation buffer (1× PBS pH 7.4, 1% formaldehyde and 0.1% protease inhibitor cocktail) for 10 min. The reaction was halted with 10× glycine to a final concentration of 0.125 M for 5 min. Tissue was pelleted at 2,000 rpm for 5 min and washed with cold PBS twice. The pellet was resuspended in cold cell lysis buffer (10 mM Tris pH 8, 10 mM NaCl, 3 mM MgCl₂, 0.5% NP40) for 15 min. Nuclei were pelleted at 2,000 rpm for 5 min at 4°C and resuspended in nuclear lysis buffer (1% SDS, 5 mM EDTA, 50 mM Tris pH 8). Chromatin was prepared and quantitated as described previously (Green et al., 2017 (link)). Briefly, ChIP was performed with 4 µg of each antibody H3K9K14ac (cat # 9441, Cell Signaling), Nurr1 (cat # sc-991, Santa Cruz), Pitx3 (cat # 19307, Santa Cruz), Normal Rabbit IgG (cat# 12-370, Millipore) overnight at 4°C. Isolated DNA was subjected to qPCR to determine relative enrichment of histone and transcription factor proteins in DAT promoter regions. Gapdh levels were measured as a control using the same assay methods and antibodies. Primer sequences for qPCR were obtained from a previously published manuscript (He et al., 2011 (link)) and are found in Table 1. For each ChIP assay, data were normalized to input, IgG and represented as enrichment relative to control.