Tgx fastcast acrylamide gel

Samples were lysed in M-PER mammalian protein extraction reagent (Thermo Fisher Scientific) supplemented with a proteinase inhibitor cocktail (Nacalai Tesque) and phosphatase inhibitor PhoSTOP (Roche). They were separated using TGX FastCast acrylamide gels (Bio-Rad Laboratories, Hercules, CA, USA) and transferred using a Trans-Blot Turbo Transfer System (Bio-Rad Laboratories). The membranes were blocked with 5% skim milk in Tris-buffered saline (150 mM NaCl and 20 mM Tris–HCl, pH 7.2) for 1 h at room temperature and then incubated with primary antibodies overnight at 4 °C. The membranes were then treated with horseradish peroxidase-conjugated donkey anti-rabbit secondary antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature. Each membrane was treated with WB Stripping Solution Strong (Nacalai Tesque) and stained with anti-actin beta antibody (MilliporeSigma), followed by incubation with horseradish peroxidase-conjugated donkey anti-mouse IgG secondary antibody (1:1000; Santa Cruz Biotechnology). The bound antibodies were visualized using SuperSignal West Pico (Thermo Fisher Scientific) on an Image Quant LAS 4010 imager (GE Healthcare Life Sciences, Pittsburgh, PA, USA). ACTB was used as an internal control.

Liver tissues were homogenized in a solution containing T-PER solution and 1× HALT protease inhibitor cocktail (40 μl/mg tissue). Homogenates were further diluted in T-PER to a concentration of 1 mg/ml protein with 2x Laemmli buffer. Protein samples (20–30 μg) were subjected to Western Blot analysis. Thus, proteins separated by electrophoresis in 10% TGX FastCast acrylamide gels (Bio-Rad) were transferred to PVDF membrane (Bio-Rad, TurboBlot system) and probed with rabbit polyclonal anti-Arg1 (C-terminal peptide; 1:10,000 dilution; Abcam ab91279) and mouse polyclonal anti-β-actin (1:7500 dilution; Sigma) or anti-tubulin antibodies. Immunoreactive proteins were detected using HRP-conjugated goat anti-rabbit or anti-mouse secondary antibody (1:7500; Sigma) enhanced chemiluminescence signal. Digitized images were recorded with a FluorChem 8900 instrument (Alpha Innotech, San Leandro, CA). Some images underwent semi-quantitation with publicly available Image J software (Version 1.47, NIH).

The dentate gyrus part of the hippocampus was dissected and homogenized in RIPA lysis buffer with protease inhibitor and phosphatase inhibitor cocktails (Sigma-Aldrich, St. Louis, MO, USA) using TissueLyser II (Qiagen, Hilden, Germany). Protein concentration was measured with a BCA Protein Assay Kit (Pierce, Appleton, WI, USA). Proteins were separated and transferred using 10% TGX FastCast Acrylamide Gels and Trans-Blot Turbo Mini PVDF Transfer Packs (Bio-Rad, Hercules, CA, USA). Western blot was performed according to a standard procedure, with anti-CD44 antibody (R&D Systems, Minneapolis, MN, USA, AF6127; 1:2500) and anti-GAPDH antibody (Abcam, Cambridge, UK, ab9485; 1:5000), with the latter being used as a loading control. For band visualization, the chemiluminescence detection method was used. For densitometric calculations, a scan of the X-ray film was analyzed using the Band/Peak Quantification plugin [50 ] in Fiji 2.3.0 software [29 (link)] and normalized to GAPDH.

Total proteins in cells or tissues were extracted with cold lysates and separated by 10% TGX FastCast acrylamide gel (Bio-Rad, United States), followed by being transferred to 0.45 μm PVDF membranes (Millipore, United States). Subsequently, the PVDF membrane was sealed with a blocking solution for 15 min at room temperature. The appropriate dilution antibody (FOXP1, CST, Catalog No. D35D10, United States) and anti-GAPDH antibody (Proteintech, Catalog No. 10494-1-AP, United States) were applied for overnight incubation on a shaker in a 4°C fridge. After washing, the secondary antibody was applied for 1 h incubation. The chemiluminescence (Thermo) fluid was applied for exposure. Quantity One software (Bio-RAD) read the protein bands and interpreted the patterns into numeric data.

Briefly, myocardial tissue protein extracts prepared from control and treated samples in different groups were suspended in PBS containing protease inhibitor cocktail, and 50 μg of protein was loaded onto 10 % TGX FastCast Acrylamide gel (Bio-Rad Laboratories Ltd). Electrophoresis, immunoblotting, and protein detection were done for α-smooth muscle actin (α-SMA) using anti α-SMA (Sigma Adrich) antibody. Bands were visualized with Fluor S-MultiImager MAX system (Bio-Rad Laboratories, Canada) and quantified by image analysis software (Quantity One, Bio-Rad Laboratories, Canada).

Vero cells were seated on a 12-well plate at a cell density of 2 × 10⁵ cells/well. Cells were infected with SARS-CoV-2 at an MOI of 1 and incubated at 37°C for 24 h. The cultured cells were subjected to centrifugation at 1,600 × g for 5 min. The supernatant was removed, and the cells were lysed using 1 × SDS sample buffer. The lysed cells were boiled at 95°C for 3 min, sonicated using Bioruptor II (BM Equipment, Tokyo, Japan), and centrifuged at 16,400 × g for 3 min. The samples were loaded onto a 10% TGX FastCast Acrylamide gel (Bio-Rad, CA, USA) and electrophoresed. Then, the proteins were transferred to a polyvinylidene difluoride membrane (Millipore). The membrane was blocked with PBS containing 0.05% tween-20 (PBS-T; Nacalai Tesque) and 5% skimmed milk (Nacalai Tesque) and reacted with rabbit anti-SARS-CoV-2 S1 (clone: SA39; Cell Engineering Corporation, Osaka, Japan) or mouse anti-S2 monoclonal antibodies (GeneTex, CA, USA). The membrane was washed with PBS-T twice and reacted with HRP-conjugated anti-mouse or rabbit IgG antibodies (Sigma-Aldrich). Protein bands were visualized using Chemi-Lumi One Ultra (Nacalai Tesque) and Amersham ImageQuant 800 (GE Healthcare, IL, USA).