Enhanced chemiluminescence reagent

Cells were lysed with buffer containing 1% sodium dodecyl sulfate (SDS) (Sigma, St. Louis, MO, USA), 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, and cOmplete EDTA-free protease inhibitor cocktail (11873580001; Roche Diagnostics, Mannheim, Germany). Total cell lysates were denatured in 6× Laemmli’s buffer and boiled for 5 min. Samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad Laboratories, Hercules, CA). Membranes were blocked with a mixture of 5% milk with Tris-buffered saline–Tween 20 (TBST) and incubated overnight at 4°C with primary antibodies. Membranes were then washed with TBST and incubated with appropriate horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Membranes were washed again with TBST, and bands were visualized with enhanced chemiluminescence reagent (advansta, Menlo Park, CA, USA).

Total protein was abstracted from NPTr cells which were collected and lysed with Mammalian Protein Extraction Reagent (Cwbio, Beijing, China) after transfection and/or infection. Then, the concentration of total protein was measured using Bradford Protein Assay Kit (Beyotime, Shanghai, China) following the manufacturer’s instructions. Equal protein quantities were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to the nitrocellulose membrane (GE Life Science, Piscataway, NJ, USA). The membranes were blocked in 5% bovine serum albumin (BSA) for 1 h and then incubated with specific primary antibodies for 2 h at room temperature. After being washed three times, the membranes were incubated with relative second antibodies for 1 h. Finally, the protein blots were visualized using enhanced chemiluminescence reagent (Advansta, Menlo Park, CA, USA). The primary antibodies against β-actin and Horseradish peroxidase-conjugated anti-mouse/rabbit secondary antibodies were purchased from Abclonal Technology (Wuhan, China). Rabbit polyclonal antibody against viral NP was purchased from GeneTex, Inc. (San Antonio, TX, USA).

RAW264.7 cells (1 × 10⁶ cells/dish in 6-cm dish) were co-treated with LPS with or without various doses of GTEO (25–100 μg/mL) for various time points. After treatment, cells were detached and washed in cold PBS twice. Then, they were lysed in a radioimmunoprecipitation assay (RIPA) buffer (Pierce Biotechnology, Rockford, IL, USA). The concentrations of proteins were determined using a Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein samples (60–100 mg/well) along with sample dye were denatured for 5 min at 94 °C. SDS-PAGE was used to separate the protein samples, followed by overnight transfer onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 0.1% Tween-20 in PBS containing 5% non-fat skim milk for 30 min at room temperature, reacted with primary antibodies for 2 h, and then incubated with either HRP-conjugated goat anti-rabbit or anti-mouse antibodies for 1 h. An enhanced chemiluminescence reagent (Advansta, Inc., San Jose, CA, USA) was used to develop the immunoblots, images were captured by the ChemiDoc XRS⁺ docking system, and the protein bands were quantified by using Imagelab software (Bio-Rad Laboratories, Hercules, CA, USA).

Human OS tissue and cell lines were lysed in RIPA lysis buffer (Cat# P0013B, Beyotime) supplemented with a 1× protease inhibitor cocktail (Cat# P1005, Beyotime) for 30 min at 4°C, followed by a 15-min centrifugation step at 12,000 × g to generate supernatants. Then, a bicinchoninic acid protein assay kit (Cat# ab102536, Abcam) was used to determine protein concentrations. Next, 5× protein loading buffer (Cat# P0015; Beyotime) was added to lysates and denatured for 5 min at 100°C. Approximately 20 μg protein was separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene fluoride membranes. Membranes were then blocked in 5% fat-free milk for 45 min at room temperature, followed by an overnight incubation with primary antibodies at 4°C. Next, membranes were rinsed three times in 1× TBST buffer and further incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 60 min at room temperature. Protein signals were determined using enhanced chemiluminescence reagent (Cat# K-12045-D10, Advansta, San Jose, CA, USA). Antibodies were rabbit anti-Mettl3 (Cat# 15073-1-AP, Proteintech, Wuhan, China), rabbit anti-β-actin (1:3,000, Cat# 4,970, Cell Signaling, Danvers, MA, USA), and goat anti-rabbit IgG H&L (HRP) (1:1,500, Abcam, Cat# ab205718).

Fresh cells were lysed on ice in radioimmunoprecipitation assay buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate and 1 mM phenylmethylsulfonyl fluoride) containing protease and phosphatase cocktail inhibitors (Cat. HY-K0010 and Cat. HY-K0021; MedChemExpress) for 30 min. The lysates were then cleared by centrifuging at 12,000 g for 10 min. The protein concentration was quantified using the Bradford assay. Protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane (Millipore, Burlington, MA, USA), and immunoblotted with specific antibodies. Protein expression was detected using a ChemiDoc XRS Imaging System (Bio-Rad Laboratories; Hercules, CA, USA) with an enhanced chemiluminescence reagent (Advansta, San Jose, CA, USA).

After treatment with FA-1 (150 nM) and exposure to CSE, we harvested cell lysate with 1X RIPA buffer (Sigma-Aldrich), including protease and phosphatase inhibitor (Thermo Fisher). Protein concentration was determined with BCA protein assay reagent (Thermo Fisher). Protein samples were separated with SDS-PAGE and transferred to polyvinylidene fluoride membrane. The membrane was blocked with 5% non-fat milk in TBS with 0.5% Tween-20, and incubated with primary and second antibodies, and visualized with enhanced chemiluminescence reagent (Advansta). The approximate positions (kDa) of prestained protein standard (Bio-Rad) are indicated on the right of the blots. Equivalent loading was verified by stripping the blot and reprobing with antibodies to β-actin. Relative quantification of signal intensity was determined using image Lab software (Bio-Rad, Hercules, CA, USA) and expressed as a relative densitometry.