Ab23832

Nicotine in drinking water (Sigma, N-3876; 200 μg/mL) contains 1% saccharin (Shyuanye, 128-44-9). Ordinary feed: composed of corn meal, flour, wheat husk, vitamins, soybean meal, oil residue, fish meal, cod liver oil, stone powder, and salt. Vitamin D3 special feed: vitamin D3 (10,000 U/Kg) was added to ordinary feed. Previously, vitamin D3 supplementation with 10,000 IU QD improved the anxiety and depression scores in patients with Crohn’s Disease [11 (link)]. α7 nAChR (a subunit of neuronal nicotinic acetylcholine receptors) primary antibody (ab23832, 1:1,000), and Goat anti-rabbit IgG H&L (ab6721, 1:10,000) which was the secondary antibody came from Abcam (Cambridge, United Kingdom). NR2A primary antibody (19953-1-AP, 1:800) and GAPDH primary antibody (10494-1-AP, 1:10,000) were purchased from proteintech. 5× All-In-One RT MasterMix kit (Bioland Scientific LLC, FS01-01) and GoTaq^® qPCR Master Mix (promega, A6020) were used for cDNA synthesis. The gene-specific primers, NR2A, α7 nAChR, and GAPDH, were purchased from Sangon Biotech (Shanghai, China).

Cells were washed twice with cold 1xPBS, scraped off the plates, collected by centrifugation for 5 minutes at 6,000rpm, and lysed in M2 lysis buffer (20mM Tris-HCl pH6.0, 0.5% NP-40, 250mM NaCl, 3mM EGTA, and 3mM EDTA) containing protease inhibitors as described in our previous work [38 (link)]. After lysis, protein concentration was measured using Bradford assay (BioRad) and equal amounts of proteins were resolved on 8% SDS-page polyacrylamide gels, and transferred onto nictrocellulose membranes using BioRad semi-dry transfer unit. Membranes were blocked using 5% nonfat dry milk in 1xPBS containing 0.1–0.5% Tween20. After rinsing with 1xPBST, membranes were incubated overnight at 4 degrees Celsius with 2.5μg/mL α7 nAChR rabbit polyclonal (Abcam ab23832 and ab10096) or 1:25,000 β-actin mouse monoclonal (Sigma-Aldrich A1978) primary antibodies, washed again in 1xPBST, incubated for one hour at room temperature in HRP-conjugated secondary antibodies at 1:3000 dilutions, and protein was detected using ECL reagent from GE Healthcare or Pierce Biotechnology according to standard protocols. Results were quantitated using ImageJ software where protein expression was normalized to the corresponding β-actin, and represented as fold change relative to the control as graphical data.

Total protein content was measured using a DC Protein Assay Kit (Biorad, Hercules, CA). Amounts of lysates containing equal protein content were then diluted in loading buffer (120 mM Tris, 20% (v/v) glycerol, 10% (v/v) mercaptoethanol, 4% (w/v) SDS, 0.05% (w/v) bromophenol blue, pH 6.8), incubated for 10 minutes at 95°C, submitted to gel electrophoresis in AnykD gel (Biorad), and blotted onto polyvinylidene fluoride membranes (BioRad). Membranes were washed in Tris-buffered saline with 0.1% Tween 20 (TBST) and blocked in TBS containing 5% (w/v) dry milk powder, which was also used for antibody incubations. Incubation with primary antisera directed against β2 (1:1,000, provided by Dr. Cecilia Gotti), α3, α4, α5, α6, 5-HT₃ (1:100 #sc-1771, sc-5591, sc-28795, sc-27292, sc-28958 Santa Cruz Biotechnology), α7, β4 (1:1,000 #ab23832 and 1:100 #ab156213 Abcam, Cambridge, UK) was performed overnight at 4°C on parafilm in a humidified container, followed by 3×10 minute washes in TBST and 1 h incubation at 21°C with horseradish peroxidase-conjugated secondary antibody (1:2,000, Dako, Glostrup, Denmark). After thorough washing in TBST, enhanced chemiluminescence Western blotting detection reagents (Western Lightning ECL Pro, Perkin Elmer, Waltham, MA) were used for signal detection and protein bands were visualized using a Chemidoc XR digital image analyser (Biorad).