Rabbit anti gapdh polyclonal antibody

After 48-72 hours of relevant treatment, the cultured cells were washed twice with PBS (phosphate-buffered solution), and then, 50-100 µl RIPA lysis buffer (Sangon Biotech, Shanghai, China) supplemented with PMSF (phenylmethane sulfonyl fluoride), phosphatase and protease inhibitor (KeyGEN BIOTECH, Nanking, China) was used to elute the total protein. After measurement of protein concentration with a BCA (bicinchoninic acid) kit (Merck Millipore, Darmstadt, Germany), 50-100 µg of total protein was loaded and separated on a 10% SDS polyacrylamide gel, transferred to a PVDF (polyvinylidene difluoride) membranes, and incubated with anti-p21, anti-p27, anti-CDK2, anti-phospho-c-Jun or anti-phospho-Smad2 monoclonal rabbit antibody (1:1000, CST, Danvers, MA, USA); anti-E-cadherin, anti-β-catenin, anti-vimentin, anti-snail, anti-slug, anti-c-Jun, anti-Smad2, anti-CREB1, anti-JNK1 or anti-GAPDH rabbit polyclonal antibody (1:1000-1:5000, Proteintech, Chicago, IL, USA); or anti-CCAR1 rabbit polyclonal antibody (1:1000, SAB, Baltimore, MD, USA) overnight at 4°C. Protein bands were visualized using a chemiluminescence kit (Merck Millipore, Darmstadt, Germany).

A549R cells were seeded into 10 cm tissue culture dishes (Corning), incubated for 24 h, and treated with complex 4 for 12 h. The cells were washed with ice-cold PBS and lysed by incubation in radio immune precipitation assay buffer (RIPA) and a protease inhibitor cocktail (Sigma) for 30 min on ice. The lysates were centrifuged at 15000 rpm for 15 min at 4 °C, and the protein concentrations were quantified by a BCA protein assay reagent kit (Sigma). The proteins were separated on precast NuPAGE 4% to 12% polyacrylamide gradient Bis–Tris gels (Invitrogen) under denaturing conditions, transferred to PVDF membranes (Invitrogen), and subjected to Western blot analyses. Thioredoxin reductase antibody (Proteintech USA) and rabbit anti-GAPDH polyclonal antibody (Proteintech USA) were diluted (1:2000, respectively) in PBS containing 5% nonfat powdered milk and 0.1% Tween-20 and incubated with the membrane overnight at 4 °C. Horseradish peroxidase conjugated secondary antibodies (Proteintech USA) were used. The bound immune complexes were detected using an ECL prime Western blot detection reagent (Amersham Inc., USA). The images were captured on FluorChem M (ProteinSimple, Santa Clara, CA).

PBMCs were treated with FIN and cells were harvested at different time points and lysed with lysis buffer for the detection of phosphorylation of transcription factors, phosphatase inhibitor was added into the lysis buffer. The lysate were then subjected to electrophoresis, followed by transferring onto the membrane and detection with the primary antibodies including rabbit anti-STAT1 polyclonal antibody (abcam, The United Kingdom), rabbit anti-STAT6 polyclonal antibody (ABclonal, Baltimore, MD), rabbit anti-phospho STAT1 (Tyr 701) monoclonal antibody (Cell Signaling Technology, Danvers, MA), rabbit anti-phospho STAT6 (Tyr 641) polyclonal antibody (ABclonal), rabbit anti-phospho NF-κB p65 (Ser536) monoclonal antibody (Cell Signaling Technology), rabbit anti-COX2 polyclonal antibody (R&D systems), rabbit anti-nuclear matrix protein p84 monoclonal antibody (abcam), rabbit anti-β-Actin polyclonal antibody (Proteintech) and rabbit anti-GAPDH polyclonal antibody (Proteintech).

For cell lysates, transfected 293 cells were washed and lysed in cell lysis buffer (Cell Signaling). Lysates were then diluted with 4 × sample buffer (Biorad) and boiled for 10 min before subjected to western blotting. For virions, clarified supernatants were loaded onto a thin layer of 8.4% optiprep density gradient medium (Sigma-Aldrich) and placed in a TLA55 rotor (Beckman Coulter) for ultracentrifugation for 2 h at 20,000 rpm. The pellet was then resuspended for western blotting. For protein detection, the following antibodies were used: rabbit anti-SARS-CoV-2 S monoclonal antibody (Thermofisher), mouse anti-SARS-CoV-2 S1 (R&D systems), rabbit anti-GAPDH polyclonal antibody (Proteintech), horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse IgG polyclonal antibody (Cell Signaling). Chemiluminescence was detected using ChemiDoc Touch Imaging System (Bio-Rad). The cleavage ratio of S1 or S2 to FL was determined by densitometry using ImageJ (NIH).