Western blotting analysis was conducted as previously reported [40 (link)]. Anti-Runx2 (Cat No. WL03358, 1:500, WanleiBio, Shenyang, China), anti-MMP9 (Cat No. WL03096, 1:500, WanleiBio, China), anti-MMP13 (Cat No. WL04694, 1:500, WanleiBio, China), anti-Col10a1 (Cat No. AF6538, 1:500, Beyotime, Shanghai, China), anti-Caspase 3 (Cat No. WL02117, 1:500, WanleiBio, China), anti-Cleaved caspase 3 (Cat No. WL01992, 1:500, WanleiBio, China), anti-β-actin (Cat No. 20536-1-AP, 1:1000, Proteintech, Wuhan, China) primary antibodies were used.
Anti caspase 3
Manufactured by Wanlei
Sourced in China
Anti-caspase-3 is a laboratory reagent used in research applications. It is an antibody that specifically binds to and detects the caspase-3 protein, which is involved in the process of programmed cell death or apoptosis. The core function of Anti-caspase-3 is to enable the identification and quantification of caspase-3 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti cleaved caspase 3, by Wanlei (2 mentions)
Anti bax, by Wanlei (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Anti mmp9, by Wanlei (1 mentions)
Anti runx2, by Wanlei (1 mentions)
Anti mmp13, by Wanlei (1 mentions)
Anti β actin, by Proteintech (1 mentions)
Anti p21, by Abcam (1 mentions)
Anti p akt, by Cell Signaling Technology (1 mentions)
β actin, by Bioss Antibodies (1 mentions)
Anti p27, by Abcam (1 mentions)
Anti akt, by Proteintech (1 mentions)
Thymol, by Merck Group (1 mentions)
Anti beclin1, by Abcam (1 mentions)
Hrp labeled goat anti mouse igg h l, by Beyotime (1 mentions)
Anti iκb α 4814, by Cell Signaling Technology (1 mentions)
Anti phospho ampkα thr172, by Cell Signaling Technology (1 mentions)
Anti phospho mtor ser2448 d9c2, by Cell Signaling Technology (1 mentions)
Anti cleaved caspase 9, by Wanlei (1 mentions)
Transwell system, by NEST Biotechnology (1 mentions)
5 protocols using anti caspase 3
1
Western Blot Analysis of Osteoarthritis Markers
2
Quantitative Protein Analysis in Tumor Cells
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HeLa and CaSki cells and nude mouse tumor tissues were lysed by RIPA lysis buffer containing 1% PMSF and 1% protein phosphatase inhibitor for 30 min and then centrifuged at 14,000 × g at 4°C for 20 min. Collecting the supernatant to store at −80°C. The protein concentration was performed by Bradford assay kit. Cells and tissue lysates were electrophoresed by SDS-PAGE and proteins were transferred to PVDF membrane (Millipore) and then blocked with Tris-buffered saline Tween-20 with 5% non-fat milk for 2 h. Next, they were incubated with primary antibodies diluted with TBST overnight at 4°C, the primary antibodies were as follows: Anti-hnRNP A2/B1 (Bioworld Technology, St. Louis Park, MN, USA; 1:1,000), anti-AKT (Proteintech, Wuhan, China; 1:500), anti-p-AKT (Cell Signaling Technology, Danvers, MA, USA; 1:1,000), anti-p21 (Abcam, Cambridge, UK; 1:2,000), anti-p27 (Abcam; 1:1,000), anti-PI3K (Wanleibio, Shenyang, China; 1:500), anti-caspase-3 (Wanleibio; 1:500), anti-cleaved caspase-3 (Wanleibio; 1:500), β-actin (Bioss, Beijing, China; 1:6,000) was used as a loading control and followed by incubation with secondary antibody (ZSGB-Bio, Beijing, China; 1:90,000) at room temperature for 1 h. Chemiluminent detection was determined by ECL kit. ImageJ software was used to conclude data.
3
Molecular Mechanisms of Anti-inflammatory Effects
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The thymol (purity > 98.5%), LPS (Escherichia coli 055:B5, L2880), and dimethyl sulfoxide (DMSO, D4540) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The mouse monoclonal anti-β-actin (AA128), HRP-labeled goat anti-rabbit IgG (H + L), and HRP-labeled goat anti-mouse IgG (H + L) antibodies (A0208, A0216) were purchased from Beyotime (Shanghai, China). The following antibodies were used in this work: anti-IκB α (4814, Cell Signaling Technology, Danvers, MA, USA), anti-phospho-NF-κB p65 (Ser536, 3033S, Cell Signaling Technology), anti-NLRP3 (AF2155, Beyotime), anti-IL-1β (AF7209, Beyotime), anti-beclin1 (ab231341, Abcam), anti-ATG7 (AA820, Beyotime), anti-LC3B (ab229327, Abcam, Cambridge, UK), anti-phospho-AMPK-α (Thr172, Cell Signaling Technology), anti-phospho-mTOR (Ser2448, D9C2, Cell Signaling Technology), anti-caspase-3 (WL02117, Wanleibio, Shenyang, China), and anti-cleaved caspase-9 (WL01838, Wanleibio).
4
Overexpression of SPINK1 Regulates Cell Proliferation and Apoptosis in Hepatocellular Carcinoma
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The HepG2, Hep3B, H22, and 293T cell lines were given as gifts by Professor Aiguo Wang (Dalian Medical University, Dalian, China), who purchased them from the Chinese Academy of Sciences, Beijing, China. The cells were grown in McCoy’s medium (Sangon Biotech, Shanghai, China), RPMI-1640, or Dulbecco’s modified Eagle’s medium (DMEM) (Sangon Biotech, Shanghai, China) with 10% fetal bovine serum (FBS) (CellMax, AusGeneX, Queensland, Australia). Human SPINK1-overexpressing (OE) and SPINK1 short hairpin RNA (shRNA) lentiviral vectors were generated by Applied Biological Materials (ABM, Nanjing, China), and the Transwell system was purchased from Nest (Southborough, MA, USA). The following commercially available antibodies were used: anti-SPINK1 (Abnova, Taipei, Taiwan) and anti-Bcl-2, anti-Bcl-XL, anti-Bax, anti-Bad, and anti-caspase-3 (all from Wanlei Biotech, Shenyang, China). The main chemicals or reagents used in this study were as follows: 5-fluoruracil (5-FU) (Shanghai Pharmaceutical Company, Shanghai, China), Lipo2000 (Invitrogen, Carlsbad, CA, USA), Cell Counting Kit-8 (CCK-8) (Xian Baiying Biotechnology Inc., Xian, China), puromycin (Solarbio, Beijing, China), and polybrene (Maokang Biotech, Shanghai, China).
5
Protein Extraction and Western Blot Analysis
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Cells were harvested and lysed in radio immunoprecipitation assay (RIPA) buffer (Solarbio, China) supplemented with phenylmethylsulfonyl fluoride (PMSF) (a serine protease inhibitor, Solarbio, China) for 30 min. BCA kit (Solarbio, China) was used to determine the concentration of protein in the supernatant. Proteins were separated on a 10% SDS-PAGE gel and transferred to a PVDF membrane (Merck Millipore, USA), and then incubated overnight with primary antibody (4 °C) after being blocked for 2 h in nonfat milk solution (5%). The membrane was reacted with TBST-diluted HRP-conjugated secondary antibody for 2 h at room temperature. Protein bands were detected by Amersham Imager 600 System (General Electric Company, USA) using ECL Substrate (Beyotime Biotech, China), and quantified with Image J software. The primary antibodies contain anti-GAPDH (BBI, China), anti-GLUD1 (BBI, China), anti-E-cadherin (1:1000, Abcam, USA), anti-Vimentin (1:1000, Boster, China), anti-Cytochrome C (ProteinTech, USA), anti-BCL2 (ProteinTech, USA), anti-BAX (Wanleibio, China), anti-Caspase 3 (Wanleibio, China), anti-JNK (Wanleibio, China), anti-phospho-JNK (Wanleibio, China), anti-p53 (Cell Signaling Technology, USA), anti-p38 (Cell Signaling Technology, USA) and anti-phospho-p38 (Cell Signaling Technology, USA).
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