Sds page sample loading buffer

Cells were lysed using a Cell Lysis Buffer for Western and IP (Beyotime, P0013), followed by centrifugation to remove cell debris. The lysates were then mixed with SDS-PAGE Sample Loading Buffer (Beyotime, P0015) and heated at 100°C for 5 minutes to denature the proteins. SDS-PAGE gels (12.5%) were prepared using the SDS-PAGE Gel Quick Preparation Kit (Beyotime, P0012AC). Protein transfer was conducted using the Turbo Transfer System (BIO-RAD). The following antibodies were used: IFIT1 Polyclonal Antibody (Proteintech, #23247-1-AP), IFIT2 Polyclonal Antibody (Proteintech, #12604-1-AP), IFIT3 Polyclonal Antibody (Proteintech, # 15201-1-AP), IFIT5 Polyclonal Antibody (Proteintech, # 13378-1-AP), GFP Tag Monoclonal Antibody (Proteintech, #66002-1-Ig), Myc Tag Mouse Monoclonal Antibody (Beyotime, #AF0033), Flag Tag Mouse Monoclonal Antibody (Beyotime, #AF2852), GAPDH Mouse Monoclonal Antibody (Beyotime, #AF0006), and Actin Mouse Monoclonal Antibody (Beyotime, #AA128). LSDV074 (H3L) Mouse Monoclonal Antibody, an intermediate/late protein commonly used for capripoxvirus detection [36 (link),57 ], was generously provided by Dr. Chunsheng Yin (China Institute of Veterinary Drug Control, IVDC). Rabbit anti-LSDV serum was prepared in our laboratory using LSDV virus inactivated at 65°C and mixed with an immune adjuvant (Biodragon, cat#KX0210045). Western blotting analyses were repeated three times and band densities were measured and analyzed using ImageJ software, with GAPDH or Actin serving as the normalization reference.

Total protein was extracted from cells with RIPA lysis buffer (P0013C, Beyotime) containing phosphatase inhibitor (C0002, TargetMol) and protease inhibitor (C0001, TargetMol), and then the protein quantity was determined using BCA Protein Assay Kit (P0012, Beyotime). The samples were prepared using SDS‐PAGE Sample Loading Buffer (P0015L, Beyotime). Subsequently, the samples were denatured at 105 °C for 15 min. For immunoblot assay, immunoprecipitates or whole‐cell lysates were loaded to SDS‐PAGE, typically with a load of 30 µL (30‐40 µg). Then, the proteins in the gel were transferred onto PVDF western blotting membranes and then blotted as described previously.^[52 (link),
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RNA was extracted from heart tissue using the TRIZOL extraction method. To obtain purity and integrity of RNA, OD₂₆₀/OD₂₈₀ and OD₂₆₀/₂₃₀ of RNA were detected using Nano Drop (Nano Drop technologies, Wilmington, NC, USA), and 18S rRNA and 28S rRNA bands of RNA were observed using electrophoresis (1.2%). The cDNA was obtained using a PrimeScript RT reagent kit (Takara, Dalian, China, cat. No. RR047A) according to the manufacturer’s instructions. The primers for the selenoprotein, inflammation, apoptosis, and necroptosis related genes, as well as the housekeeping gene GAPDH, are shown in Table 1. All primers were designed using Oligo 7 (DBA Oligo, Inc., Cascade, CO, USA). The PCR products were run in an agarose gel to ensure the presence of a single specific amplification product and confirm specificity of RT-PCR. The melting curves had sharp, single peaks with the expected TM values. The cDNA templates were amplified using 2× SYBR Green qPCR Master Mix reagents (Bimake, Shanghai, China, cat. No. B21203) and gene expression was detected using the Roche LightCycler 480II system (Basel, Switzerland). A two-step method was used in the reaction process, including pre-denaturation at 95 °C for 5 min; amplification with 40 cycles, each cycle including 95 °C for 15 s and 60 °C for 40 s; and a melting reaction. Three replicate wells were set up for each sample to ensure the accuracy of the results. The 2^−ΔΔCt method was used for quantification analysis.
Minced myocardium (approximately 50 mg) was homogenized in 500 μL of RIPA lysis buffer (Beyotime bio, Shanghai, China, cat. No. P0013B)/PMSF (Beyotime bio, Shanghai, China, cat. No. ST506) mixture (100:1) using an Ultra-Turrax homogenizer at low temperature. The homogenates were centrifuged at 12,000 rpm for 10 min at 4 °C, and the resultant supernatants were collected. The total protein concentration was calculated with a BCA Protein Assay Kit, then diluted in SDS-PAGE sample loading buffer (Beyotime bio, Shanghai, China, cat. No. P0015L) and boiled for 15 min. The final concentration of the protein sample was 3 μg/μL. Equal amounts of protein (30 μg protein/line) were loaded and separated by 6–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis depending on the size of the target protein. After electrophoresis, the proteins were transferred to 0.2 μm nitrocellulose filter membrane (PALL, New York, NY, USA, cat. No. 66485), blocked in 5% (w/v) non-fat milk or 10% (w/v) BSA for 2 h and incubated overnight at 4 °C with the primary antibodies. Primary antibodies used in this experiment as follow: GPX1 (WL02497a), β-tubulin (WL01931), COX-2 (WL01750), iNOS (WL0992a), Bax (WL01637), Bak (WL0129a), Bcl-2 (WL01556), Caspase-3 (WL02117), Caspase-7 (WL02360), Caspase-8 (WL03426), Caspase-9 (WL03421), XIAP (WL03561), APAF1 (WL04536), TNF-α (WL01581), TNFR1 (WL01414), TRAF2 (WL02846), IKK α/β (WL01900), P-IκBα (WL02495), P-P50 (WL01866), P65 (WL01980), P-P65 (WL02169), P-ERK1/2 (WLP1512), P-JNK (WL01813), P-P38 (WLP1576), cIAP1 (WL03666), cIAP2 (WL01254), and c-FILP (WL02485) were purchased from Wanleibio (Shenyang, China); HIF-1α (BS-0737R), RIPK1 (BS-5805R), and RIPK3 (BS-3551R) were purchased from Bioss (Beijing, China); Txnrd3 (19517-1-AP) and MLKL (21066-1-AP) were purchased from Protintech (Rosemont, IL, USA); GAPDH (AC001) was purchased from ABclonal (Wuhan, China); Cleaved Caspase-3 (9661T) was purchased from Cell Signaling Technology (Beverly, MA, USA); GPX4 (ab41787) was purchased from Abcam (Cambridge, UK). β-tubulin or GAPDH was chosen as the internal reference according to the protein size, respectively. The membrane was washed in TBST and incubated with Goat Anti-Rabbit IgG (H + L) (Abclonal, Wuhan, China, cat. No. AS014) for 2 h at room temperature. Immuno-reactive bands were visualized with a ECL luminescence reagent (Meilunbio, Dalian, China, cat. No. MA0186-1). Protein bands were photographed using a Tanon-5200 gel imaging system. ImageJ (NIH, Bethesda, MD, USA) was used to analyze the blot signal and density.

Lung tissue homogenate and macrophages were harvested, and proteins were extracted using RIPA buffer (Beyotime) containing protease inhibitors cocktail (Roche, Mannheim, Germany). To concentrate supernatants for western blot, 700 μL 100% methanol and 175 μL trichloromethane were added to 700 μL supernatant and vortexed for 30 s. Supernatants were then centrifuged at 13000 rpm for 5 min at 4°C. The supernatant liquid was removed, and added 700 μL 100% methanol, then centrifuged at 13000 rpm for 5 min at 4°C. Supernatants were discarded. And the remaining pellet was resuspended in 20 μL10% SDS, then added 4 μL 5×SDS-PAGE sample loading buffer (Beyotime) and boiled for 10 min at 95°C. The protein concentrations were measured with Pierce™ Rapid Gold BCA Protein Assay Kit (Thermo Fisher Scientific, Grand Island, USA).
Equal amounts of protein or all protein from supernatants were subjected to 8%-12% gradient polyacrylic amide gel under reducing conditions. Gels were transferred into polyvinylidene difluoride membranes (Millipore, USA), blocked with 5% fat-free milk or 5% albumin from bovine serum (BSA, Biofroxx, Germany) at room temperature for 1.5 h. The blots were reacted with the primary antibody at 4 °C overnight, followed by horseradish peroxidase-conjugated secondary antibody (1:1000; Cell Signaling Technology, USA), and detection by ChemiDoc XRS (Bio-Rad, USA). The intensities of the bands were quantified using the Image Lab Analyzer software (Bio-Rad). β-actin, α-tubulin, or GAPDH were used as a loading control. The antibodies used in the study are shown in Table 1.