Monoclonal anti β actin

Cells lysates were prepared as previously described^{17 ,62 (link)} followed by western blots using the following antibodies: mouse anti-SARS-CoV/SARS-CoV-2 S A-19 (1: 5000, GeneTex), mouse anti-SARS CoV/SARS-CoV-2 ORF7a 3C9 (1: 1000, GeneTex), mouse anti-V5 (1: 5,000, Thermo Fisher Scientific), rat anti-MLV p30 R187 (1:1000, ATCC, CRL-1912), rabbit anti-HA C29F4 (1: 2,000, Cell Signaling Technology), mouse anti-HA 2-2.2.14 (1: 5000, Invitrogen), rabbit anti-FLAG D6W5B (1: 2,000, Cell Signaling Technology), rabbit anti-GAPDH 14C10 (1: 3000, Cell Signaling Technology), rabbit anti-SARS-related Coronavirus Nucleocapsid protein 019 (1: 2,000, BEI Resources, NIH, NIAID, NR-53793), mouse anti-HIV-1 p24 AG3.0 (1: 2000, NIH/AIDS Reagent Program, ARP-4121), monoclonal anti-β-actin (1: 7,000, Sigma-Aldrich). Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1: 2,000, Cell Signaling Technology), HRP-conjugated anti-rat IgG (1: 2000, Cell Signaling Technology) and HRP-conjugated anti-mouse IgG (1: 7,000, EMD Millipore) were used for detection using the enhanced chemiluminescence detection kits Clarity and Clarity Max ECL (Bio-Rad). Quantitation of bands in western blots were performed using the ImageJ software (National Institutes of Health; https://imagej.nih.gov/ij/).

Cells were harvested and lysed with lysis buffer at various duration post transfection. Cell lysis buffer consisted of 150 mM sodium chloride, 1.0% NP-40, 50 mM Tris pH 8.0) supplemented with protease inhibitor cocktail (Roche). The whole cell lysate (20 µg) were resolved on 10% polyacrylamide sodium dodecyl sulphate gels and analysed by immunoblotting technique with sheep anti-CDy (PA185365, ThermoFisher Scientific) and monoclonal anti-β-Actin (A2228, Sigma-aldrich), respectively.

We determined the effect of SPLP ingestion on hepatic antioxidant enzyme expression. We used 0.5 g of liver to discern the possible molecular mechanism after SPLP ingestion with Western blotting. Briefly, the homogenization, extraction, and quantification of hepatic proteins were performed. The primary antibodies raised against sheep anti-CuZnSOD (superoxide dismutase, CuZn, Oxis Health products, Inc.), sheep anti-MnSOD (superoxide dismutase, Mn: Oxis Health products, Inc.), anti-human erythrocyte catalase (Assay Designs, Inc.), sterol regulatory element-binding protein-1 (SREBP-1, Santa Cruz Biotechnology, Inc.), and monoclonal anti-β-actin (Sigma-Aldrich, Inc.) were used. Proteins on SDS-PAGE gels were transferred to nitrocellulose filters and stained. The density of the band with the appropriate molecular mass was semi-quantitatively determined with densitometry by using an image analysis system (Alpha Innotech, San Leandro, CA, USA).

The total proteins were prepared from cultured chondrocytes obtained as in ‘Quantitative analysis of gene expression by qRT‐PCR’ by lysis in the suspension of the RIPA Lysis and Extraction Buffer (Thermo Scientific) using Sonics Vibra Cell™ (Sonics & Materials, Newtown, CT, USA). Total cellular proteins were quantitatively analysed using the bicinchoninic acid (BCA) total protein quantitation assays 38. Equal proteins were separated by 8.0% SDS‐PAGE, blotted onto nitrocellulose membrane (Bio‐Rad, Hercules, CA, USA). The membranes were blocked by skim milk powder solution in Tris‐buffered saline and then incubated with diluted primary antibodies of CTNNB1 (AVIVA, San Diego, CA, USA) against β‐catenin, COL2A1 (Santa Cruz) against COL II by specifically binding to the C‐terminal epitope of a highly conserved motif between human and rabbit, and COL1A (COL‐1; Santa Cruz) against COL I, respectively, with the Monoclonal Anti‐β‐Actin (Sigma‐Aldrich) against the house‐keeping gene β‐actin used as an internal control. The binding proteins were detected with conjugated secondary antibody, goat anti‐rabbit IgG‐HRP (Santa Cruz) and visualized using Clarity™ Western ECL Substrate Kit (Bio‐Rad). The images were captured and analysed using the Image Lab (Beta 1) Version 3.0.1 Changelist 40296 software associated with Molecular Imager^®, ChemiDoc™ XRS+ Imaging System (Bio‐Rad).