Cellular protein was lysed by radio immune precipitation assay (RIPA) buffer (Beyotime, China) with phenylmethane sulfonyl fluoride (PMSF) protease inhibitor (Beyotime, China) and the homogenate was centrifuged at 12,000 × g for 5 min at 4 ◦C. The supernatant was collected and the protein concentration was determined immediately using a BCA protein quantification kit (Beyotime, China). The proteins were separated in 10% SDS-PAGE and transferred onto PVDF membrane, and then probed with antibodies following standard procedures. The antibodies and their dilutions utilized for western blots were as follow: anti-GHR (bs-0654R; Bioss, China; 1:500), anti-PGC1α (bs-1832R; Bioss, China; 1:500), anti-NRF1 (12,482–1-AP; Proteintech, USA; 1:500), anti-TOMM20 (AF1717; Beyotime, China; 1:500), anti-JAK2 (bs-0908R; Bioss, China; 1:1000), anti-p-JAK2 (bsm-52171R; Bioss, China; 1:1000), anti-AKT1 (bs-0115 M; Bioss, China; 1:500), anti-p-AKT1 (66,444–1-Ig; Proteintech, China; 1:500), anti-CREB1 (bs-0035R; Bioss, China; 1:500), anti-p-CREB1 (bs-0036R; Bioss, China; 1:500), anti-β-actin (bs-0061R; Bioss, China; 1:1000), goat anti-rabbit IgG-HRP (bs-0295G; Bioss, China; 1:5000), goat anti-mouse IgG-HRP (bs-0296G; Bioss, China; 1:5000).
Bs 0296g
Manufactured by Bioss Antibodies
Sourced in China
The Bs-0296G is a lab equipment product manufactured by Bioss Antibodies. It is designed for general laboratory use. The core function of the Bs-0296G is to provide a tool for researchers and scientists in their work.
Automatically generated - may contain errors
Lab products found in correlation
Bs 0295g, by Bioss Antibodies (5 mentions)
Culture cell total protein extraction reagent, by Boster Bio (2 mentions)
Rabbit anti β actin, by Proteintech (2 mentions)
Bs 0061r, by Bioss Antibodies (2 mentions)
Protease inhibitor pmsf, by Beyotime (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Bca protein quantification kit, by Beyotime (1 mentions)
Anti p akt1, by Proteintech (1 mentions)
Anti β actin bs 0061r, by Bioss Antibodies (1 mentions)
Goat anti mouse igg hrp, by Bioss Antibodies (1 mentions)
Anti pgc1α bs 1832r, by Bioss Antibodies (1 mentions)
Il 1β, by Proteintech (1 mentions)
Tnf α, by Bioworld Technology (1 mentions)
Ab184247, by Abcam (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Interleukin 6 (il 6), by Cell Signaling Technology (1 mentions)
P mtor, by Cell Signaling Technology (1 mentions)
Tissue mitochondria isolation kit, by Beyotime (1 mentions)
Protein extraction kit, by BestBio (1 mentions)
P62 18420 1 ap, by Proteintech (1 mentions)
10 protocols using bs 0296g
1
Western Blot Analysis of Cellular Signaling
2
Mitochondrial Protein Extraction and Analysis in Cerebral Infarction
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Total proteins of infarcted cortex were extracted using the protein extraction kit (BB-3101, Bestbio), and mitochondrial proteins of infarcted cortex were isolated using Tissue Mitochondria Isolation Kit (C3606, Beyotime). The concentration of proteins was measured using the bicinchoninic acid method, and loading buffer was added to obtain equal concentrations (3 μg/μL). Samples were subjected to 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred on a 0.45 μm polyvinylidene difluoride membrane. After washing with tris-buffered saline with 0.1% Tween® 20, the membrane was sealed using a rapid sealing solution for 20 min and then incubated with primary antibodies at 4°C overnight, and the corresponding secondary antibodies (bs-0296G, Bioss) were incubated for 2 h at room temperature.
The following primary antibodies were used: LC3B (L7543, Sigma), p62 (18420-1-AP, Proteintech), mTOR (#2983, Cell Signaling Technology), p-mTOR (#5536, Cell Signaling Technology), IL-1β (16806-1-AP, Proteintech), IL-6 (#12153, Cell Signaling Technology), TNF-α (BS1857, Bioworld Technology) and β-actin (#3700, Cell Signaling Technology), COX IV (ab202554, Abcam), Drp1 (ab184247, Abcam), and Parkin (JF82-09, Novus).
The following primary antibodies were used: LC3B (L7543, Sigma), p62 (18420-1-AP, Proteintech), mTOR (#2983, Cell Signaling Technology), p-mTOR (#5536, Cell Signaling Technology), IL-1β (16806-1-AP, Proteintech), IL-6 (#12153, Cell Signaling Technology), TNF-α (BS1857, Bioworld Technology) and β-actin (#3700, Cell Signaling Technology), COX IV (ab202554, Abcam), Drp1 (ab184247, Abcam), and Parkin (JF82-09, Novus).
3
Protein Extraction and Western Blot
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The protein lysate was obtained from culture cells by culture cell total protein extraction reagent (BOSTER, Wuhan, CHN) with protease inhibitor and phosphatase inhibitor cocktail. Protein concentration was determined by a BCA protein analysis kit (BIOMED, Beijing, CHN). Primary antibodies included rabbit anti-β-actin (#20536-1-AP; Proteintech, Wuhan, CHN), rabbit anti-ZO-1 (#21773-1-AP; Proteintech, Wuhan, CHN), rabbit anti-Occludin (#27260–1-AP; Proteintech, Wuhan, CHN), rabbit anti-Claudin 1 (#bs-1428R; Bioss, Beijing, CHN), rabbit anti-NF-κB P65 (#bs-0465R; Bioss, Beijing, CHN) and rabbit anti-phospho-NF-κB p65 (#3033 T; CST, Danvers, MA USA). Goat anti-rabbit (#bs-0296G; Bioss, Beijing, CHN) was used as secondary antibody.
4
Western Blot Analysis of Liver Proteins
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Liver samples were homogenized in lysis buffer [10 mmol/L Tris, pH 7.5, 150 mmol/L NaCl, 1% Triton X-100, 1 mmol/L phenylmethylsulfonyl fluoride, 0.2 mmol/L sodium orthovanadate, 0.5% Nonidet P-40 containing protease and phosphatase inhibitor cocktail tablets (Roche) at 4°C for 30 minutes. Protein was quantified using the protein assay (#21011, Intron, Korea), and proteins were resolved on 7.5%-10% SDS/ PAGE gels. The membranes were blocked for 1 hour in tris-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T) and 5% skim milk and then incubated overnight at 4°C with a primary antibody (see above) in the same buffer. The blots were then washed three times in TBS-T for 15 minutes to remove excess antibody, and then the membranes were incubated for 2 hours with secondary antibodies in TBS-T+ 5% (g/vol) skim milk: (AE-1475 Bioss, Wobrun, MA, USA) or anti-mouse (bs-0296G). Following three washes in TBS-T, proteins were detected with ECL solution (XLS025-0000, Cyanagen, bologna, Italia) and Chemi Doc (Fusion Solo, Vilber Lourmat, France).
5
Protein Extraction and Western Blot Analysis
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The total protein of each group was extracted by a protein extraction kit (KGP250, KeyGEN, China) and quantified by a protein quantification kit (KGP902, KeyGEN, China). The protein sample size was 20 µg. The total protein was obtained by SDS‒PAGE (P1200, Solarbio, China), and the protein was transferred to solid-phase carrier PVDF membrane (IPFL00010, Millipore, Germany), and the primary antibody was added: anti-RAGE (ab216329, abcam, UK), anti-HMGB1 (6893, CST, USA), anti-IL-17RA (bs-2606R, Bioss, China), anti-TAK1 (ab109526, abcam, UK), anti-Phospho-TAK1 (bs-5435R, Bioss, China), anti-β-actin (bs-0061R, Bioss, China), incubated overnight at 4 °C, added secondary antibody: Goat anti-rabbit (bs-0295G, Bioss, China), Goat anti-mouse (bs-0296G, Bioss, China), incubated at room temperature for 40 min and added substrate (WBKLS0100, Millipore, Germany), developed by chemiluminescent image (A300, Azrue, USA).
6
Protein Expression Analysis in Cells
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Cells were lysed in RIPA buffer. Protein concentration was measured using a BCA protein assay kit (Thermo Scientific, USA). Equal amounts of cell lysates were subjected to SDS–PAGE, transferred to PVDF membranes, blocked in 5% BSA, incubated with primary antibody overnight and visualized using ECL Detection Reagents (Pierce, USA). Images were acquired using a LAS-4000 Imager (Fuji). Antibodies used include mouse antibodies to β-actin (BOSTER #BM0627, 1:200) and rabbit antibodies to E-cadherin (BOSTER #PB0583, 1:200), Vimentin (BOSTER #PB0378, 1:200), and TET1 (Santa Cruz #sc-163443, 1:200) and HRP anti-rabbit and HRP anti-mouse (Bioss #bs-0295G and bs-0296G, 1:5000).
7
Immunofluorescence Analysis of Vascular Smooth Muscle
8
Western Blot Analysis of Necroptosis Signaling
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Tissues and cells were lysed using RIPA lysis buffer (Beyotime Biotechnology) containing protease and phosphatase inhibitor, homogenated, and centrifuged. The protein concentration was determined by BCA protein assay (Beyotime Biotechnology). Equal concentrations of proteins were separated in a 10% SDS-PAGE and transferred to PVDF membrane. The membrane was blocked using 5% milk in TBST, probed with primary antibody, RIPK1 (17519-1-AP, Proteintech, 1:1000), MLKL (GTX107538-100, GeneTex, 1:1000), RIPK3 (17563-1-AP, Proteintech, 1:1000), p-MLKL (EPR9514, Abcam, 1:1000), PKA (bs-0520R, Bioss, 1:1000), p-PKA (#9621, Cell signaling, 1:1000), CREB (AF6188, Affinity, 1:1000), p-CREB (AF3189, Affinity, 1:1000), P2Y14R (bs-12028R, Bioss, 1:1000), GSDMD (ab209845, Abcam, 1:1000), Caspase1 p20 (bs-10442R, Bioss, 1:1000), Bcl-2 (bs-0032R, Bioss, 1:1000), Bax (AF0120, Affinity, 1:1000), Caspase-3 p-17 (Cell Signaling Technology, #9664, 1:1000), β-actin (bs-0061R, Bioss, 1:1000), GAPDH (BS-2188R, Bioss, 1:1000) and corresponding HRP-conjugated antibodies Goat Anti-Rabbit IgG H&L Antibody (bioss, Bs-0295G, 1:8000) or Goat Anti-Mouse IgG H&L Antibody (bioss, Bs-0296G, 1:8000). Densitometry was quantified using ImageJ software.
9
Cardiac Fibroblast Protein Analysis
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Total protein was extracted from cardiac fibroblasts and myocardial samples. The proteins were separated and transferred to polyvinylidene fluoride (PVDF) membranes. Then, the PVDF membranes were blocked for 2 h at room temperature and incubated overnight at 4°C with one of the following primary antibodies: anti-collagen II (Abcam, ab188570, UK), anti-SMA (Abcam, ab7817, UK), PI3K (Abcam, ab191606, UK), p-PI3K (Abcam, ab182651, UK), AKT (Abcam, ab179463, UK), p-AKT (Abcam, ab192623, UK), mTOR (Abcam, ab134903, UK), p-mTOR (CST, 5536, USA), GAPDH (Bioss, bs-0755 R, China), goat anti-mouse (Bioss, bs-0296 G, China), or goat anti-rabbit (Bioss, bs-0295 G, China). The procedures were performed as previously described, and protein expression was examined by a chemiluminescence analyzer (Azure, A300, USA).
10
Protein Extraction and Western Blot Analysis
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The protein lysate was obtained from tissue by culture cell total protein extraction reagent (BOSTER, Wuhan, CHN) and mammalian tissue protein extraction reagent (BOSTER, Wuhan, CHN), with protease inhibitor and phosphatase inhibitor cocktail, respectively. Protein concentration was determined by a BCA protein analysis kit (BIOMED, Beijing, CHN). The primary antibodies included the following: rabbit anti-β-actin (#20536-1-AP; Proteintech, Wuhan, CHN), rabbit anti-ZO-1 (#21773-1-AP; Proteintech, Wuhan, CHN), rabbit anti-occludin (#27260-1-AP; Proteintech, Wuhan, CHN), rabbit anti-Claudin 1 (#bs-1428R; Bioss, Beijing, CHN), rabbit anti-NF-κB P65 (#bs-0465R; Bioss, Beijing, CHN), rabbit anti-phospho-NF-κB p65 (#3033T; CST, Danvers, MA USA), rabbit anti-p38 (#ab27986; Abcam, Cambridge, MA, USA), and rabbit anti-phospho-p38(#ab47363; Abcam, Cambridge, MA, USA). The secondary antibody used was goat anti-rabbit (#bs-0296G; Bioss, Beijing, CHN).
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