Bca protein concentration test kit

Total proteins were extracted from the L₄–L₆ spinal cord segment and BV2 cells in different groups using the RIPA lysis buffer(containing 1 mM phenyl-methylsulfonyl fluoride and protease inhibitor), and the protein concentration was measured using the bicinchoninic acid (BCA) protein concentration test kit (Beyotime, Shanghai, China). Equivalent protein amounts were analyzed on 11% and 8% SDS–PAGE and transferred onto polyvinylidene fluoride (PVDF, Millipore) membrane. The membranes were blocked in non-fat dried milk solution(5% in phosphate-buffer saline) at room temperature for 1.5 h, and then incubated at 4 ℃ overnight with primary antibodies. After washing with 1XTBST, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h. Bands were visualized with electrochemiluminescence detection reagents. Band intensity was quantified using the Image-Pro Plus 6.0 (Media Cybernetics, Rockville, MD, USA). The detailed information of antibodies used in this study is provided in Additional file 2: Table S2.

Dimethyl sulfoxide (DMSO), 2′,7′-dichlorfluorescin diacetate (DCFH–DA), and 2,2-azobis (2-amidinopropane) dihydrochloride solution (ABAP) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Epigallocatechin gallate (EGCG, HPLC grade, purity > 98%) and kaempferol (HPLC grade, purity > 98%) were obtained from Solarbio Science & Technology Co., Ltd. (Beijing, China). EGCG and kaempferol were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mg/mL and then freshly diluted in culture medium. The final concentration of DMSO in culture medium was below 0.05% (v/v). Phosphate buffer solution (PBS), minimum Eagle’s medium (MEM), fetal bovine serum (FBS), 0.05% trypsin–EDTA solution, penicillin (10,000 u/mL), and streptomycin (10,000 μg/mL) were purchased from HyClone (Logan, UT, USA). The Cell Counting Kit-8 (CCK-8) was obtained from Dojindo China Co., Ltd. (Shanghai, China). The BCA protein concentration test kit; cell lysis buffer for Western and IP; and phenylmethanesulfonyl fluoride (PMSF, 100 mM), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) assay kits were purchased from Beyotime Biotechnology (Shanghai, China). All other chemicals and reagents with analytical grade used in this study were obtained from Sinopharm Group Chemical Reagents Co. Ltd. (Beijing, China).

HEK 293 T cells were cultured for 24 h, and transfected with the plasmid pcDNA3.1-EGFP-Flag-CD2v-Flag or infected with 1 × 10⁶ TCID₅₀ of PRV-ΔgE/ΔgI/ΔTK-(CD2v). Samples were collected 48 h after transfection or viral infection. The protein concentration was determined by a BCA protein concentration test kit (from Beyotime Biotechnology, Shanghai, China)). Sixty μg of proteins were loaded for SDS–polyacrylamide gel electrophoresis and transferred to the PVDF membrane. The membrane was sealed with 5% skim milk at room temperature for 2 h, then incubated with the mouse anti-Flag monoclonal antibody (1:1000 dilution, ProteinFind® Anti-DYKDDDDK Mouse Monoclonal Antibody HT201-01 (TransGen Biotech, (Beijing, China)) or GAPDH (14C10) Rabbit mAb (Cell Signaling Technology, USA) in 5% skimmed milk plus 100 mg/L NaN₃ at 4 °C for 8 h, washed with TBST buffer for 3 times, followed by incubation with secondary antibody IRDye 800cw donkey anti-mouse IgG (LI-COR) (1:10,000) at room temperature for 2 h. The membrane was washed with TBST and visualized by using LI-COR Odyssey infrared fluorescence scanning imager.