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Colonic ME and MS samples were homogenized on ice in RIPA buffer (Cell signaling, Danvers, USA) supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail 2 and 3 (Sigma-Aldrich, St. Louis, USA). After centrifuging at 12,000 g at 4°C for 15 minutes, the supernatant was collected and resolved by a standard immunoblotting method. Equal quantities (20~100 μg) of total protein were run on pre-made 4–12% Bis-Tris SDS-PAGE (Invitrogen, Carlsbad, CA). OPRM1 (MOR-1) antibody (1:2000) was from Thermo-fisher scientific (Austin, TX) (Cat# PA3–203, Lot# ra224670). OPRD1 (DOR-1) antibody (1:500) was purchased from Life span biosciences (Seattle, WA, USA) (Cat# LS-C383200, Lot# 126591). OPRK1 (KOR-1) antibody (1:200) was purchased from Santa Cruz biotechnology (Dallas, TX, USA) (Cat# Sc-374479, Lot# K2816). β-actin antibody (1:5000, Sigma, St. Louis, USA) was used as loading control. The protein band detection was performed using Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE).

Dorsal spinal cord tissues at the L5 and L6 levels were removed, dissected, and homogenized in 300 μl RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM Na₃VO₄, and 1 mM NaF in the presence of the protease inhibitor cocktail. Samples were then put on ice for 30 min with shaking. Lysates were centrifuged at 13,000 g for 30 min at 4°C and the supernatant was collected. The protein concentration was quantified using a DC protein assay kit (Bio-Rad, Hercules, CA). Thirty μg of total proteins of each sample was loaded and separated on 4–12% Bis-Tris SDS-PAGE (Invitrogen). The resolved proteins were transferred to nitrocellulose membranes. The membranes were treated with 5% bovine serum albumin in Tris buffer containing Tween 20 for 2 h and then incubated with a rabbit anti-nNOS antibody (Cat. #07-571-I, EMD Millipore) overnight at 4°C. The membrane was washed three times and then incubated with horseradish peroxidase–conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch, West Grove, PA) for 1 h at room temperature. The protein band was revealed with an ECL Plus detection kit (ThermoFisher, Rockfort, IL), and the protein band intensity was quantified by using the ImageJ software program. The amounts of proteins were normalized by GAPDH (Cell Signaling Technology), which was used as a protein loading control.

Histone H3 methylation assays were conducted by incubating 5 µM MLL1 or MLL1 variant with 0.5 µM ³H-AdoMet (Perkin Elmer, Inc.) and 100 µM H3 peptide for 8 hours at 15° C. Reactions were quenched with SDS loading buffer, separated by 4–12% Bis-Tris SDS PAGE (Invitrogen) in MES buffer at 200 volts for 30 minutes. Coomassie brilliant blue stained gels were photographed and soaked for 30 minutes in autoradiography enhancer solution (Enlightning, PerkinElmer, Inc.), dried for 2 hours at 72°C under constant vacuum and exposed to film (Kodak BioMax MS Film) at −80° C for 16–18 hours.