Duox2

Flash-frozen mouse ileum was resuspended in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 5 mM EDTA, 0.1% SDS), containing protease inhibitors (Roche, cat. #: 11836170001, South San Francisco, CA, USA). Tissue was homogenized and then centrifuged at 12,000× g for 15 min, after which supernatants were collected. Protein concentration was determined using the Protein Assay Dye Reagent (Bio-Rad, cat. #: 500-0006, Hercules, CA, USA). 40 µg protein mixed with 4× Laemmli sample buffer were incubated at 37 °C for 15 min and subjected to SDS-PAGE. After overnight transfer onto PVDF membrane (Bio-Rad, cat. #: 1620177, Hercules, CA, USA), membrane was incubated overnight with primary antibody at 4 °C. Membrane was washed and then incubated with secondary antibody for 1 h, followed by application of chemiluminescent reagent ECL (GE Healthcare, cat. #: RPN2232, Chicago, IL, USA) and imaging on the ChemiDoc Imaging System (Bio-Rad), after which relative protein concentration was determined by densitometry. The primary antibodies used were DUOX2 (Santa Cruz, cat. #: sc-398681, Dallas, TX, USA), GAPDH (Santa Cruz, cat. #: sc-32233, Dallas, TX, USA). The secondary antibody was mouse IgG kappa binding protein conjugated to horseradish peroxidase (Santa Cruz, cat. #: sc-516102, Dallas, TX, USA).

The cells in different groups were lysed in lysis buffer supplemented with protease and phosphatase inhibitors (USA, B14001 and B15001) and underwent centrifugation. Protein concentrations were determined using a BCA protein assay kit (Bio-Rad, Hercules, CA, USA), and equal amounts of protein were analyzed by SDS-PAGE on 8% gels. The gels were transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore Corporation, Billerica, MA, USA). After blocking for 2 h using 5% skimmed milk, the membranes were incubated at 4°C overnight with primary antibodies DUOX2 (Santacruz, CA, USA, sc-398681; 1 : 200 dilution) and GAPDH (Santacruz, CA, USA, sc-25778; 1 : 1000 dilution). Following washing with TBST, the membranes were incubated with HRP-conjugated secondary antibodies (1 : 10000 dilution) at 37°C for 2 h and then analyzed. Western blotting results were independently performed thrice.

Total protein samples were isolated from frozen liver tissue using RIPA lysis buffer, containing protease and phosphatase inhibitor cocktail (TransLab, #30-04CLI19SSH). Samples were separated in a 10% SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membrane (GE Healthcare Life Sciences, #10600023). After the membranes were blocked in 5% skim milk for 1 h at room temperature, they were incubated with primary antibodies overnight at 4 °C and then with the corresponding secondary antibodies for 1 h at room temperature. All of the primary antibodies gp91-phox antibody (Santa Cruz Biotechnology, #K0817) and β-actin (Cell Signaling, #4970 s) were used at a dilution of 1:1000 except DUOX1 (Santa Cruz Biotechnology, #B2817) (1:500) and DUOX2 (Santa Cruz Biotechnology, #D0317) (1:500). Secondary antibodies were used at 1:2500 dilution. Immunoreactive bands were detected using the enhanced chemiluminescence (ECL) detection system with a PhosphorImager (GE Healthcare). Protein expression levels were normalized to the levels of the β-actin, which was used as a loading control.