Western bright peroxide

A peptide library of the LpqH protein sequence consisting of 15-mer peptides with 10-mer overlap was created (Genescript Inc). 4 µg of each peptide was spotted on to a nitrocellulose membrane. Once the membrane was dry, it was blocked with 5% skim milk in 1X TBST for 1 h followed by probing with 10 µg/mL of HuMab-28-009 (human IgG1) in block solution at 4 °C overnight. The blot was then washed and probed with anti-human IgG (1:1000) in block solution for 1 h. The blot was finally visualized by chemiluminescence method of detection, with substrate Western Bright ECL (Advansta, United States, Cat. No. R03031-D25) and Western Bright peroxide (Advansta, United States, Cat. No. R03025-D25) in a 1:1 ratio for 15 s exposure time. The blots were washed with 1X TBST five times after primary and secondary antibody incubation. Blot image shown in Supplementary Fig 4 is derived from the same experiment, and they were processed in parallel (Data availability—Figshare 10.6084/m9.figshare.23550108).

A peptide library of the LpqH protein sequence consisting of 15-mer peptides with 10-mer overlap was created (Genescript Inc). 4 µg of each peptide was spotted on to a nitrocellulose membrane. Once the membrane was dry, it was blocked with 5% skim milk in 1X TBST for 1 h followed by probing with 10 µg/mL of HuMab-28-009 (human IgG1) in block solution at 4 °C overnight. The blot was then washed and probed with anti-human IgG (1:1000) in block solution for 1 h. The blot was finally visualized by chemiluminescence method of detection, with substrate Western Bright ECL (Advansta, United States, Cat. No. R03031-D25) and Western Bright peroxide (Advansta, United States, Cat. No. R03025-D25) in a 1:1 ratio for 15 s exposure time. The blots were washed with 1X TBST five times after primary and secondary antibody incubation. Blot image shown in Supplementary Fig 4 is derived from the same experiment, and they were processed in parallel (Data availability—Figshare 10.6084/m9.figshare.23550108).

Total cell extracts were prepared in lysis buffer containing 2% SDS, 62.5 mM Tris pH 6.8, 10% glycerol, 5% β-mercaptoethanol. Protein samples were heated at 95 °C for 10 min and analyzed by SDS-PAGE on 8% or 12% gels (45 min, 200 V) (Bio-rad Laboratories), followed by immunoblotting on PVDF membranes using a wet blotting system (70 min, 250 mA) (BioRad Laboratories). Membranes were incubated with primary and secondary antibody diluted in 5% milk/TBST solutions, Western blots were developed using Western Bright peroxide (Advansta, 180129-34, CA, USA) and visualized on ChemiDoc XRS+ system (Bio-Rad, CA, USA).

Human neuroblastoma cells line SK-N-SH (ATCC® HTB­11™) were purchased from American Type Culture Collection (ATCC, USA). Minimum essential medium (MEM), fetal bovine serum (FBS), penicillin-streptomycin, Trypsin, and phosphate buffered saline (PBS) solutions were obtained from Gibco Life Technologies (Invitrogen, USA). Dimethyl sulfoxides (DMSO), retinoic acid (RA), thiazolyl blue tetrazolium bromide (MTT), and forskolin were purchased from Sigma-Aldrich (USA). Morphine sulphate pentahydrate (M-35-SU) and d,I-methadone.HCl (MET-637) were purchased from Lipomed AG (Switzerland). Isobutylmethylxanthine (IBMX) and radioimmunoprecipitation assay (RIPA) buffer and protease inhibitor were purchased from Amresco, USA. The antibodies used, α-synuclein, calmodulin, synaptotagmin 1, VAMP 2, anti-β-actin, and horseradish peroxidase (HRP) were purchased from Cell Signaling Technology (Massachusetts). WesternBright™ ECL and WesternBright Peroxide were purchased from Advansta (USA).

A peptide library of the LpqH protein sequence consisting of 15-mer peptides with 10-mer overlap was created (Genescript Inc). 4 µg of each peptide was spotted on to a nitrocellulose membrane. Once the membrane was dry, it was blocked with 5% skim milk in 1X TBST for 1 h followed by probing with 10 µg/mL of HuMab-28-009 (human IgG1) in block solution at 4 °C overnight. The blot was then washed and probed with anti-human IgG (1:1000) in block solution for 1 h. The blot was finally visualized by chemiluminescence method of detection, with substrate Western Bright ECL (Advansta, United States, Cat. No. R03031-D25) and Western Bright peroxide (Advansta, United States, Cat. No. R03025-D25) in a 1:1 ratio for 15 s exposure time. The blots were washed with 1X TBST five times after primary and secondary antibody incubation. Blot image shown in Supplementary Fig 4 is derived from the same experiment, and they were processed in parallel (Data availability—Figshare 10.6084/m9.figshare.23550108).

Dimethyl sulphoxide (DMSO), molecular biology grade, used as a solvent for all stocks of chemical agents, was obtained from Roth (Karlsruhe, Germany). All reagents used in flow cytometry analysis were purchased from BD Biosciences Pharmingen (San Diego, CA, United States). The following primary antibodies were used: anti-GAPDH (Merck Millipore, #MAB374, 1:20000, 30 min, RT), anti-p-p65 (Ser529) (Biorbyt, # orb 14916, 1:500, overnight, +4°C), anti-DHFR (BD Biosciences), and anti-TS (Merck Millipore, #MAB4130, 1:500, overnight, +4°C). Secondary goat anti-rabbit IgG-HRP (Dako, #P0448, 1:2000, 1h, RT) and anti-mouse IgG-HRP (Dako, #P0447, 1:1000, 1h, RT) were used. Hoechst 33342 (Life Technologies, #H3569) and anti-mouse Alexa Fluor 555 were used in the IF study. Protease inhibitors (#11 836 153 001) were from Roche Applied Science (Mannheim Germany). The nitrocellulose membrane was from GE Healthcare Life Sciences (Freiburg, Germany), solvents for HRP reaction (Western Bright Peroxide and Western Bright Quantum) were purchased from Advansta, ECL reagent was from Millipore (United States), and CX-4945 was obtained from Biorbyt. Other solvents, reagents, and chemicals were purchased from POCH (Avantor Performance Materials, Gliwice, Poland), Merck, and Sigma-Aldrich Chemical Company (St. Louis, MO, United States).

Expression of ZO-1, claudin-1, occludin, nuclear factor-κB (NF-κB), and Nrf2 in the ileal mucosa of weanling pigs were measured by using Western blot analysis according to the previous study [16 (link)]. The BCA Protein Assay Kit was used to determine mucosal protein concentration. Extracts containing equal amounts of protein (30 μg) were resolved by 10% polyacrylamide gels and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membrane was blocked for 3 h with 5% skimmed milk powder and incubated overnight with antibodies (Abcam, Cambridge, UK) for ZO-1, claudin-1, occludin, NF-κB, Nrf2, and anti-β-tulubin in a 1:2000 dilution. After three washes, the secondary antibody (LI-COR, Nebraska, NE, USA) was added in a 1:10,000 dilution and incubated at 4 °C for 4 h. The membrane was washed three times and developed using WesternBright™ Peroxide (Advansta, San Jose, CA, USA) in an imaging system (Carestream, New York, NY, USA).