β actin antibody

(–)-Epigallocatechin-3-gallate (EGCG) (Selleck, China) was diluted to stock solutions of 50 mM with PBS and stored at −80°C for all subsequent experiments.
UL42, gE, and PRV-positive sera were generated in our laboratory. β-actin antibody was obtained from TransGenBiotech (Beijing, China). FITC-conjugated goat anti-pig IgG antibody was purchased from Sigma-Aldrich (St. Louis, MO). DAPI was purchased from Beyotime Biotechnology (Shanghai, China).

Samples were lysed in ice-cold RIPA buffer (0.5 M Tris-HCl, pH 7.4, 1.5 M NaCl, 2.5% deoxycholic acid, 10% NP-40, 10 mM EDTA) with protease inhibitor cocktail (Roche, Indianapolis, IN, USA). Protein quantification was performed by BCA assay, and equal amounts of protein lysate (40 μg) were separated by 10% SDS-PAGE. Transfer to nitrocellulose membranes was performed in transfer buffer (12 mM Tris base, 96 mM glycine, pH 8.3, and 15% methanol). Afterwards, the membranes were blocked for 2 hours in the TBST buffer with 5% BSA and probed with the TGF-β1, p-SMAD3, SMAD3 (all from Cell Signaling Technologies, Danvers, MA, USA; 1:500 dilution) and β-actin antibody (Transgen, Beijing, China; 1:5000 dilution) overnight at 4 °C. The membranes were washed with TBST buffer for three times, followed by incubating with appropriate HRP-conjugated secondary antibody (1:5000 dilution; TransGen Biotech Co., Ltd., Beijing, China) for 1 hour at room temperature. Finally, the protein expression levels were detected by enhanced chemiluminescence and visualized by autoradiography.

Cells were lysed in ice-cold RIPA buffer (0.5 M Tris-HCl, 10 mM EDTA, 1.5 M NaCl, 10% NP-40, 2.5% deoxycholic acid, pH = 7.4) supplemented with protease inhibitor cocktail (Roche, USA). Protein quantification was performed by BCA assay kit (TransGen Biotech, Beijing, China), and loaded samples containing equal amounts of protein (40 μg) were separated by 10% SDS-PAGE. Afterwards, the protein transfer onto nitrocellulose membranes (PALL, USA) was performed within transfer buffer (12 mM Tris base, 96 mM glycine, pH 8.3, and 20% methanol). Subsequently, the membranes were blocked in the TBST buffer containing 5% BSA for 2 h, followed by incubating with the antibody of SOX-2, OCT-4 (1:1000 dilution; all from Cell Signalling Technologies, CST, USA), RUNX-2, perilipin A, and SOX-9 (1:1000 dilution, all from BOSTER Biological Technology, Wuhan, China), and β-actin antibody (1:1000 dilution; TransGen, Beijing, China) overnight at 4 °C. After that, the membranes were washed with TBST buffer for three times, and incubated with secondary antibody (anti-rabbit HRP-conjugated; 1:8000 dilution; Santa Cruz Biotechnology) at room temperature for 1 h. Finally, the expression levels were analyzed by enhanced chemiluminescence and visualized by autoradiography.

The expression of the selected proteins in serum samples was verified by western blotting (WB), as previously reported [11 (link), 19 (link), 27 (link)]. Briefly, 30 µg of protein from each sample were separated by SDS-PAGE and transferred onto NC membranes (Millipore, Bradford, MA, USA). Then, the membranes were blocked for 2 h in PBST buffer containing 5% bovine serum albumin (BSA) and probed with IL1RL1 and THBS1 primary antibodies (1:1000 dilution, Santa Cruz Biotechnology, USA) and a β-actin antibody (1:5000 dilution, TransGen Biotech, China) at 4 °C overnight. After washing 3 times with PBST buffer for 10 min, the membranes were incubated with appropriate HRP-conjugated secondary antibodies (1:5000 dilution, TransGen Biotech, China) for 1 h at room temperature. Following another wash in TBST buffer, the protein expression levels were detected by enhanced chemiluminescence and visualized by autoradiography. Quantification of the western blot band intensity was performed using ImageJ 1.45 software (NIH) according to the manufacturer’s instructions.

EGCG was purchased from Selleck (China) and was diluted to stock solutions of 50 mm with PBS and stored at −80°C for all subsequent experiments. The purity of EGCG was 99.68% by HPLC assessed. The PEDV N antibody used herein was generated in our laboratory. The anti-PEDV-N polyclonal antibody was prepared in rabbits using PEDV-N protein as antigen and western blot and IFA were diluted 1:5,000 and 1:500, respectively (Gao, et al., 2020 (link)). The β-actin antibody was purchased from TransGenBiotech (China). HRP-labeled Goat Anti-Mouse IgG (H + L) and DAPI (4′,6-diamidino-2-phenylindole) was purchased from Beyotime Biotechnology (China). The citric acid solution (PH 3.0) is composed of 40 mm citric acid, 10 mm KCl, and135 mm NaCl, to remove un-internalized virus particles.