Hrp conjugated secondary antibody

Proteins samples were extracted from primary HSCs and LX‐2 cells using RIPA lysis buffer (Beyotime, China). Equal amounts of proteins were separated by 10% SDS‐PAGE and transferred onto PVDF membranes (Millipore, USA). The membranes were blocked with 5% skim milk for 1 hour at room temperature and then incubated with primary antibodies overnight at 4°C. The following antibodies were used for Western blotting: β‐actin (1:500; Bioss, China), Col1α1 (1:500; Bioss), α‐SMA (1:500; Bioss), PLK1 (1:1000; Abcam), Bax (1:800; Abcam), Bcl‐2 (1:800; Abcam), cleaved caspase‐3 (1:1000; Abcam), β‐catenin (1:500, Bioss), c‐Myc (1:500; Bioss) and Cyclin D1 (1:500; Bioss). The membranes were incubated with HRP‐conjugated secondary antibodies (1:10000; ZSGB‐Bio, China) for 60 minutes at room temperature. The protein bands were detected with a chemiluminescent (ECL) system (Bio‐Rad, USA) and analysed using IMAGE J software (National Institutes of Health, USA).

Proteins were obtained and homogenized in the RIPA buffer. Equivalent amounts of protein (30 μg) were loaded and separated by 10% SDS-PAGE gels, then transferred to polyvinylidene difluoride (PVDF) membranes (0.45 mm). The membranes were washed in Tris-buffered saline containing 0.05% Tween-20 (TBST) followed by blocking for 1 h using 5% nonfat milk in TBST at room temperature, then incubated at 4°C overnight with the following primary antibodies: monoclonaln antibody to Prdx6 (1:1000; ab133348, Abcam, Cambridge, MA, USA), rabbit polyclonal antibody to TLR2 and TLR4 (1:400, BA1716/BA1717, Boster Biological Technology, China), rabbit polyclonal antibody to NF-κB (1:1000; sc-109, Santacruz, CA, USA), rabbit monoclonal antibody to iNOS (1:500, bs0162R, Bioss, China), rabbit monoclonal antibody to COX-2 (1:500, 55070-1-AP, Proteintech, China,) and mouse monoclonal antibody to β-actin (1:10,000, D110007-0100, Songon Biotech, China). The next day, the blots were washed and incubated for 1 h with respective HRP-conjugated secondary antibodies (ZSGB-BIO, Beijing, China; dilution 1:3000) at room temperature. Finally, the protein was visualized using the ECL kit (Millipore, Temecula, CA, USA). The results were quantified using Quantity One 1-D analysis software, and β-actin was used as the internal control. Three independent experiments were taken.

We performed western blotting according to a standard protocol. Total cell lysates were extracted using 2% SDS lysis buffer with a protein phosphatase inhibitor mix (Applygen, Beijing), and 30 μg of total protein was prepared for electrophoresis through 8% or 12% (v/v) SDS–PAGE gels. After electrophoresis, the separated protein bands were transferred onto nitrocellulose membranes (Millipore, Ireland) and were blocked in 5% (w/v) Albumin Bovine-V (BSA-V, Solarbio, Beijing) for 1.5 h at room temperature. The primary antibodies used were as follows: anti-GAPDH (FL-335; Santa Cruz Biotechnology), anti-CD59 (HPA026494, Sigma-Aldrich), anti-CD163 (ab182422, Abcam), anti-arginase 1(Arg-1) (16001-1-AP, Proteintech), anti-IFN-γ (ab9657, Abcam), anti-iNOS (ab3523, Abcam), anti-IL-6 (ab6672, Abcam), anti-STAT3 (D1B2J, Cell Signaling Technology), and anti-phospho-Stat3 (Ser727, Cell Signaling Technology). The quality of loading and transferring was assessed by immunostaining with the anti-GAPDH antibody. The membranes were incubated with the primary antibodies at a dilution ratio of 1:1000 overnight at 4 °C. After washing three times in TBST, the membranes were incubated in 1:5000 HRP-conjugated secondary antibodies (Zsbio, Beijing) at room temperature for 1 h. Finally, the membranes were washed three times in TBST and visualized using an ECL Kit (Applygen, Beijing).

Intestinal tissue and cell protein were extracted according to the manufacturer’s instructions (KeyGEN Biotech, Nanjing, China). Western blotting was performed with primary antibodies against nurr1, p21 (Abcam, Cambridge, UK), ZO-1, occludin (Proteintech, Wuhan, China), and β-actin (ZSGB-BIO, Beijing, China) and horseradish peroxidase (HRP)-conjugated secondary antibodies (ZSGB-BIO, Beijing, China). Protein levels were analyzed using Gel-Pro Analyzer Version 4.0 (Media Cybernetics, MD, USA).

The anti-His antibody was purchased from MBL (Nagoya, Japan). Anti-MAGI3, anti-Cyclin D1 and anti-GFAP antibodies from Abcam (Cambridge, UK); Anti-β-catenin and anti-GFP antibodies from Cell Signaling Technology (Beverly, CA); Anti-Flag M2 antibody and anti-Flag M2 affinity gels from Sigma (St Louis, MO); Anti-β-actin, anti-GAPDH and HRP-conjugated secondary antibodies from ZSGB-BIO (Beijing, China).