Total protein was harvested from ox-LDL-treated hVMSCs at 24 h using RIPA lysis buffer (Thermo Scientific). The extraction (20 μg) was loaded for the standard procedures of western blot assay. The primary antibodies including proliferating cell nuclear antigen (PCNA; #2586, 1 : 2000), matrix metalloproteinase (MMP)-2 (#13132, 1 : 1000) and MMP-9 (#3852, 1 : 1000) were purchased from Cell Signaling Technology (CST; Danvers, Massachusetts, USA), and β-actin (CST; #4967, 1 : 1000) was the loading control. Blots signals were detected using an enhanced chemiluminescence (ECL) system according to the manufacturer's instructions. Image-Pro Plus (Media Cybernetics, Rockville, MD, USA) was used to quantify the densities of the protein signals. Every sample was performed western blotting in triplicate.
Matrix metalloproteinase mmp 2
Manufactured by Cell Signaling Technology
Sourced in United States
Matrix metalloproteinase (MMP)-2 is an enzyme that plays a key role in the breakdown and remodeling of the extracellular matrix. It is involved in various physiological and pathological processes, including tissue remodeling, angiogenesis, and tumor invasion.
Automatically generated - may contain errors
Lab products found in correlation
Mmp 9, by Cell Signaling Technology (7 mentions)
N cadherin, by Cell Signaling Technology (5 mentions)
E cadherin, by Cell Signaling Technology (4 mentions)
Proliferating cell nuclear antigen pcna, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Vimentin, by Cell Signaling Technology (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Cleaved caspase 9, by Cell Signaling Technology (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (2 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
Snail, by Cell Signaling Technology (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)
Image pro plus, by Media Cybernetics (1 mentions)
P γh2ax, by Cell Signaling Technology (1 mentions)
Sodium butyrate, by Merck Group (1 mentions)
Cisplatin, by Merck Group (1 mentions)
13 protocols using matrix metalloproteinase mmp 2
1
Protein Expression Analysis of ox-LDL-Treated hVMSCs
2
Western Blot Analysis of Protein Targets
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Antibodies against CHD4 (GTX124186) were purchased from GeneTex Inc. (Hsinchu City, Taiwan, R.O.C). Antibodies against Caspase 3 (#9662S), cleaved poly (ADP-ribose) polymerase (PARP) (#9541), Vimentin (#5741), N-cadherin (#5296), E-cadherin (#13116), matrix metalloproteinase (MMP)-2 (#4022), p-γH2A.X (#9718), GAPDH (#2118) and Actin (#8480) were purchased from Cell Signaling Technology (Beverly, MA, USA). Mouse (GTX213111) and rabbit (GTX213110) IgG (HRP) antibodies from GeneTex Inc. were used as the secondary antibodies.
3
Molecular Mechanisms of Sodium Butyrate and Cisplatin
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Sodium butyrate (>99% purity) and cisplatin were obtained from Sigma-Aldrich (St. Louis, MO, United States). Sodium butyrate was dissolved in ultrapure water to prepare a 900 mM stock solution and cisplatin was dissolved in absolute dimethyl sulfoxide (DMSO) to prepare a 4 mg/ml (4 mg cisplatin +1 ml DMSO) stock solution, both of which were stored at −20°C.
Rabbit monoclonal antibodies against B-cell lymphoma 2 (BCL-2), BCL2 associated X (BAX), Cytochrome C (CytC), apoptotic protease activating factor-1 (Apaf-1), apoptosis inducing factor (AIF), proliferating cell nuclear antigen (PCNA), cleaved caspase-3, cleaved caspase-9, matrix metalloproteinase (MMP)-2, MMP-9, survivin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from Cell Signaling Technology (Danvers, MA, United States). The antibodies are diluted to a working concentration at a ratio of 1:1,000 and stored at 4°C. The secondary antibodies were used at a working concentration of 1:10,000 and were obtained from LI-COR (Lincoln, NE, United States).
Rabbit monoclonal antibodies against B-cell lymphoma 2 (BCL-2), BCL2 associated X (BAX), Cytochrome C (CytC), apoptotic protease activating factor-1 (Apaf-1), apoptosis inducing factor (AIF), proliferating cell nuclear antigen (PCNA), cleaved caspase-3, cleaved caspase-9, matrix metalloproteinase (MMP)-2, MMP-9, survivin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from Cell Signaling Technology (Danvers, MA, United States). The antibodies are diluted to a working concentration at a ratio of 1:1,000 and stored at 4°C. The secondary antibodies were used at a working concentration of 1:10,000 and were obtained from LI-COR (Lincoln, NE, United States).
4
Protein Extraction and Western Blot Analysis
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Total protein of cells was extracted using lysis buffer (Pierce; Thermo Fisher Scientific, Inc.) and quantified by Bradford method. 30 µg protein was separated by SDS-PAGE. Samples were transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA) and incubated overnight at 4°C with antibody against cyclin E (20808; Cell Signaling Technology, Inc., Danvers, MA, USA), matrix metalloproteinase (MMP)2 (1:1,000; cat. no. 4022; Cell Signaling Technology, Inc.), MMP9 (1:1,000; cat. no. 3852; Cell Signaling Technology, Inc.), E-cadherin (24E10) (1:1,000; cat. no. 3195; Cell Signaling Technology, Inc.), N-cadherin (1:600; cat. no. 13116; Cell Signaling Technology, Inc.), p21 (Waf1/Cip1;12D1) (1:1,000; cat. no. 2947; Cell Signaling Technology, Inc.), p-ERK (1:1,000; cat. no. 9101; Cell Signaling Technology, Inc.), ERK (1:1,000; cat. no. 4695; Cell Signaling Technology, Inc.), β-actin (1:3,000; cat. no. 4967; Cell Signaling Technology, Inc.), GAPDH (1:3,000; cat. no. G5262; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). After incubation with peroxidase-coupled anti-mouse/rabbit IgG (1:1,000; Cell Signaling Technology, Inc.) at 37°C for 2 h, bound proteins were visualized using ECL (Pierce; Thermo Fisher Scientific, Inc.) and detected using a DNR BioImaging System (DNR, Jerusalem, Israel). Relative protein levels were quantified using ImageJ software.
5
Western Blot Analysis of SIRT1 and MMPs
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Cells specimens were lysed with RIPA lysis buffer (Boster). Protein concentrations were measured by bicinchoninic acid protein assay (Boster). Equal amounts of total protein were boiled in sample buffer and separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis.
Proteins were then transferred to a polyvinylidene fluoride membrane (Millipore) and blocked with 5% skim milk powder at room temperature for 1 hour. The membrane was incubated with rabbit monoclonal antibody against SIRT1 (#2496; Cell Signaling Technology), matrix metalloproteinase ( MMP)-2 (#40994; Cell Signaling Technology), MMP-9 (#13667; Cell Signaling Technology), and goat anti-rabbit immunoglobulin G. An electrochemiluminescence kit was used to visualize the protein bands. Protein levels were calculated relative to β-actin.
Proteins were then transferred to a polyvinylidene fluoride membrane (Millipore) and blocked with 5% skim milk powder at room temperature for 1 hour. The membrane was incubated with rabbit monoclonal antibody against SIRT1 (#2496; Cell Signaling Technology), matrix metalloproteinase ( MMP)-2 (#40994; Cell Signaling Technology), MMP-9 (#13667; Cell Signaling Technology), and goat anti-rabbit immunoglobulin G. An electrochemiluminescence kit was used to visualize the protein bands. Protein levels were calculated relative to β-actin.
6
Molecular Signaling Pathways in Cancer
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RPMI-1640 and fetal bovine serum (FBS) were obtained from Gibco-BRL (Life Technologies, Paisley, Scotland). Antibodies to total PTEN, AKT, p-AKT, BCL-2, Bax, E-cadherin, N-cadherin, Snail, matrix metalloproteinase (MMP)-2, and MMP-9 were all purchased from Cell Signaling Technology, Inc. (Danvers, CA, USA); Antibodies to Bin1 and β-actin were purchased from Abcam, Inc. (Cambridge, MA, USA). Go Taq® qPCR Master Mix was purchased from Promega (Madison, WI, USA). RevertAid™ First Strand cDNA Synthesis Kits was purchased from MBI Fermentas (Hanover, MD, USA). Annexin V fluorescein isothiocyanate (FITC) and PI double stain was purchased from BD Pharmingen (San Diego, CA, USA).
7
Evaluating EMT Markers in Ovarian Cancer
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Ovarian cancer cells were lysed using RIPA lysis buffer (Beyotime, China) with the addition of a protease inhibitor cocktail (Thermo Fisher Scientific), and total protein concentration was detected using a BCA Protein Assay kit (Pierce, Rockland, IL, USA). Extracted proteins were separated by 12% SDS-PAGE gel electrophoresis and then transferred onto polyvinylidene difluoride membranes (Roche, Basel, Switzerland). After blocking with dried skimmed milk powder dissolved in TBST solution, the membranes were incubated with specific primary antibodies against KIFC1, CENPE, E-cadherin, N-cadherin, Snail, ZEB1, matrix metalloproteinase (MMP)2, and MMP9 (Cell Signaling Technology) at 4°C overnight. Subsequently, membranes were cultured with peroxidase-conjugated secondary antibodies (Cell Signaling Technology) at room temperature for 2 h. Chemiluminescence (Millipore Corporation) and densitometry analysis by ImageJ software were applied to measure the protein expression.
8
Protein Expression Analysis in Glioma
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Protein in glioma tissues and cells were collected. The protein concentration was determined by bicinchoninic acid protein quantitative kit. The protein was boiled with 6 × loading buffer for 5 min at 95 °C. The sample was prepared and stored at − 20 °C. According to the molecular weight of the target protein, the corresponding concentration of sodium dodecyl sulfate polyacrylamide gel electrophoresis was prepared, 50 μg protein was used for electrophoresis separation, and then the protein was transferred to the nitrocellulose membrane. The membrane was blocked for 2 h, and incubated with primary antibodies Arg-1 (1:2000, Abcam, Cambridge, UK), CD163 (1:2000, Abcam), PEG3 (1:1000, Abcam), TSG101 (1:1000), CD63 (1:1000) CD81 (1:500, Santa Cruz Biotechnology, Inc, Santa Cruz, CA, USA), Bcl-2 (1:1000), Bax (1:1000), matrix metalloproteinase (MMP)-2 (1:1000), MMP-9 (1:1000, Cell Signaling Technologies, Beverly, MA, USA) and β-actin (1:3000, ZSGB-Bio, Beijing, China) overnight, then incubated with secondary antibody (1:5000, ZSGB-Bio) labeled by horseradish peroxidase for 1 h. Enhanced chemiluminescence was used for development.
9
Hesperetin and Cisplatin Combination Therapy
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Hesperetin (> 98% purity) and DDP were obtained from Sigma-Aldrich (St. Louis, MO, USA). A 200 mM stock solution of Hesperetin was prepared by dissolving it in absolute dimethyl sulfoxide (DMSO), which was stored at -20°C. A 4 mg/ml stock solution of DDP was prepared by dissolving it in normal saline, which was stored at -20°C. Lipofectamine® 2000 Transfection Reagent (Thermo Fisher Scientific, Waltham, MA, USA) was stored at 4°C. The Shanghai Genechem biotechnology company (Shanghai, China) provided plasmids PTEN-shRNA (PTEN-RNAi-38319) and PTEN-NC (negative control, CON077), which were maintained in glycerol bacterial cultures at -20°C.
Rabbit monoclonal antibodies including those recognizing PTEN, AKT, phosphorylated (p)-AKT, BCL2 associated X (BAX), B-cell lymphoma 2 (BCL2), apoptosis inducing factor (AIF), Cytochrome C (CytC), CyclinD1, matrix metalloproteinase (MMP)-2, MMP-9, cleaved caspase-3, cleaved caspase-9, caspase-9, caspase-3, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were obtained from Cell Signaling Technology (Danvers, MA, USA). The antibodies were used at a working concentration of 1:1000 and were stored at 4°C. The secondary antibodies were purchased from LI-COR (Lincoln, NE, USA) and used at a dilution ratio of 1:10,000.
Rabbit monoclonal antibodies including those recognizing PTEN, AKT, phosphorylated (p)-AKT, BCL2 associated X (BAX), B-cell lymphoma 2 (BCL2), apoptosis inducing factor (AIF), Cytochrome C (CytC), CyclinD1, matrix metalloproteinase (MMP)-2, MMP-9, cleaved caspase-3, cleaved caspase-9, caspase-9, caspase-3, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were obtained from Cell Signaling Technology (Danvers, MA, USA). The antibodies were used at a working concentration of 1:1000 and were stored at 4°C. The secondary antibodies were purchased from LI-COR (Lincoln, NE, USA) and used at a dilution ratio of 1:10,000.
10
Western Blot Analysis of Proteins in Cells
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Total proteins were extracted from cells using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). Proteins were quantified using a Pierce bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.), followed by western blot analysis. 40 µg proteins from cell lysates were subjected to 12% SDS-PAGE. Once proteins were transferred to nitrocellulose membranes, they were incubated with antibodies targeting β-actin (cat. no. 58169, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), CCR8 (cat. no. ab32131, 1:500; Abcam, Cambridge, UK), E-cadherin (cat. no. 3195, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), matrix metalloproteinase (MMP)-2 (cat. no. 40994, 1:1,000; Cell Signaling Technology, Inc.) and vascular endothelial growth factor (VEGF)-C (cat. no. 2445, 1:1,000; Cell Signaling Technology, Inc.), followed by incubation with a horseradish peroxidase-labeled goat anti-rabbit secondary antibody (cat. no. ab6721, 1:3,000; Abcam) for 1.5 h at room temperature. Protein bands were visualized by Millipore enhanced chemiluminescence (cat. no. WBKLS0500; EMD Millipore, Billerica, MA, USA). ImageJ 1.45 software (NIH) was used to perform densitometric analysis of each band.
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