Mouse anti β actin

Proteins from the colon samples were extracted in lysis buffer containing protease and phosphatase inhibitors. Protein concentrations (n = 5) were determined in the supernatant of colonic tissues by a classic BCA assay kit (Beyotime, China). Equal amounts of proteins (30 μg) were fractionated by 10% SDS-PAGE gels and transferred onto a polyvinylidene fluoride (PVDF; Millipore, USA) membrane by a Bio-Rad western blot apparatus. The membranes were blocked with 5% BSA for 1 h at room temperature and then incubated with the following primary antibodies for 24 h at 4 °C: rabbit anti-JAK2 (1:5000, Abcam, UK), rabbit anti-phospho-JAK2 (1:500–2000, Bioss, China), rabbit anti-TLR4 (1:500–1:2000, Bioss, China), mouse anti-STAT3 (1:500–1000, Bioss, China), rabbit anti-phospho-STAT3 (1:500–2000, Bioss, China), and mouse anti-β-actin (1:1000, ZSGB-BIO, China). After washing with TBST (Tris-buffered solution, pH 7.6, 0.05% Tween 20) 3 times for 10 min, the blots were incubated with anti-rabbit or anti-mouse IgG horseradish secondary antibodies (1/100,000 dilution) for 1 h at room temperature. Finally, the protein bands were visualized with ECL western blot detection reagents (Bridgen, China). The expression levels of the proteins were compared with the control based on the relative intensities of the bands.

The cells were washed three times with PBS, and protein lysate buffer containing 5 µL PMSF (1 mM) per 500 µL was placed on ice for 5 min and centrifuged at 14,000×g for 10 min after sonication. Then, the supernatant was collected. The protein concentration was measured using a bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China). After boiling with loading buffer for 10 min, equal amounts of protein (40 µg) were separated by 10% SDS–PAGE (Beyotime, Shanghai, China) and transferred onto PVDF membranes (Millipore, Massachusetts, USA). Membranes were blocked with 5% milk powder for 60 min at room temperature and incubated with specific primary antibodies at 4 °C overnight. Rabbit anti-SIRT6 (1:1000 dilution, Abcam, UK), rabbit anti-HIF-1α (1:500 dilution, Abcam, UK), rabbit anti-HK2 (1:500 dilution, Cell Signaling Technology, USA), and mouse anti-β-actin (1:1000 dilution, ZSGB-Bio, Beijing, China) antibodies were used. Proteins were detected using anti-rabbit IgG (1:2000 dilution, Cell Signaling Technology, USA) or anti-mouse IgG (1:2000 dilution, Cell Signaling Technology, USA) antibodies and a chemiluminescence detection system (FluorChem HD2, USA).

Primary antibodies used in this study included rabbit anti-SOX-2 (1:1000 dilution, Abcam, Cambridge, UK), rabbit anti-Nanog (1:1000 dilution, CST, Danvers, Massachusetts, USA), rabbit anti-OCT-4 (1:1000 dilution, Abcam), rabbit anti-pSmad2/3 (1:500 dilution, Abcam), rabbit anti-Snail (1:500 dilution, Abcam), rabbit anti-E-cadherin (1:500 dilution, Abcam), rabbit anti-TGF-β1(1:1000 dilution, Abcam), rabbit anti-N-cadherin (1:1000 dilution, Wanleibio), rabbit anti-Slug (1:1000 dilution, Wanleibio) and mouse anti-β-actin (1:1000 dilution, ZSGB-BIO, Beijing, China).

All cells were treated as indicated and lysed with lysis buffer [50 mM tris–HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% NP-40, 1 mM PMSF, and 1 × protein inhibitor (Roche)]. The cell extracts were immunoblotted with the indicated antibodies to measure the level of the expressed proteins. Mouse anti-β-actin (ZSGB-Bio), rabbit anti-GFP (Abcam), rabbit anti-PARP11 (Finetest), rabbit anti-IFNAR1(Abcam), mouse anti-poly (ADP-ribose) (GeneTex), rabbit anti ZIKV NS1 (Genetex), rabbit anti-ZIKV NS3 (Genetex), mouse anti-HA, mouse anti-His, and mouse anti-Flag tag antibodies (Sigma-Aldrich) were used for detection at the appreciated dilutions.

The glomeruli of rats were isolated using the sieving method. The glomeruli or the hepatic tissue was rinsed with ice cold 0.9% NaCl and lysed with lysis buffer (50 mM Tris, 150 mM NaCl, 1% sodium deoxycholate, 1% Triton X-100, 0.1% sodium dodecyl sulfate) containing protease and phosphatase inhibitors on ice. The lysates (100 µg) or centrifuged urine (20 µl) were denatured at 95°C for 5 min in sample buffer, separated in an 8% polyacrylamide sodium dodecyl sulfate gel, and transferred onto a PVDF membrane. The PVDF membranes were blocked at room temperature for 2 h with 5% powdered milk in Tris-HCl buffer containing 0.1% Tween 20 (TBST). Primary antibodies were diluted with TBST and added as follows: goat anti-rat Angptl4 (Santa Cruz Biotech, Delaware Avenue, CA, USA; 1∶200) and mouse anti-β-actin (Zsgb-Bio, Beijing, China; 1∶400). The membranes were incubated with primary antibodies overnight at 4°C followed by incubation with secondary antibodies (HRP-conjugated goat anti-mouse IgG and rabbit anti-goat IgG, Jackson ImmunoResearch, West Grove, PA, USA; 1∶5000) at room temperature for 1 h. There were 3 replicates for each sample. Blots were detected using a luminescent image analyzer (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and quantified using Image Quant TL (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).