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123 protocols using anti mmp2

1

Western Blot Analysis of MMPs and Collagens

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WB experimental analysis was performed according to the standard protocols. Briefly, total cell lysates were prepared in sodium dodecyl sulfate buffer. We loaded equal amounts of protein onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis for curing, and then transferred the protein to a polyvinylidene fluoride membrane. After dilution, the following primary antibody ratio was added: anti-MMP2 (Abcam, America), anti-MMP3 (Abcam, 1:2,000), anti-MMP2 (Abcam, 1:2,000), anti-gum, (Santa Cruz Biotechnology, 1:500), anti-collagen II (well, America, 1:1,000), anti-collagen I (Millip, America,1:1,000), and anti-GAPDH (Abcam, America, 1:5,000).
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2

Western Blot Analysis of Protein Markers

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Antibodies against CyclinD1, E-cadherin, β-catenin, Snail1 and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against USP42, CyclinE1, PCNA, and MMP-9 were from Abcam (Cambridge, MA, USA). Anti-MMP-2 was from Epitomics (Burlingame, CA, USA). Horseradish peroxidase-conjugated secondary antibodies were from Beyotime Biotechnology (Shanghai, China).
Cells were washed three times with PBS and then lysed in pre-cooled radioimmunoprecipitation (RIPA) assay buffer on ice for 10 min. After removal of cell debris by centrifugation (12,000g, 10 min), protein concentration of supernatants was measured by BCA protein assay kit (Thermo Fisher Scientific). After boiling for 5 min in sample buffer, equal amount of proteins of different groups were separated by SDS–PAGE, and transferred onto to a nitrocellulose membrane (Millipore, Bredford, USA). After blocking with 5% skim milk, the membranes were incubated with the primary antibodies at 4°C overnight with agitation, followed by incubation with corresponding secondary antibodies for 1 h at room temperature with agitation. Reactive protein was then detected using ECL chemiluminescence system (Bio-Rad, Richmond, CA, USA).
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3

Cab45-G Knockdown Impacts EMT Signaling

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Two Cab45-G shRNA vectors, named shCab45G-1 (GGTTCATGGTGAAGGAGAT) and shCab45G-2 (GGAAGCTGATGGTCATCTT) were obtained from Genepharma (Shanghai, China). The primary antibodies in this study were used as follows: anti-EGFP (MBL, Nagoya, Japan), anti-E-Cadherin (Cell Signaling Technologies, Danvers, MA), anti-N-Cadherin (Cell Signaling Technologies, Danvers, MA), anti-β-catenin (Cell Signaling Technologies, Danvers, MA), anti-Vimentin (Epitomics, Burlingame, CA), anti-p-ERK (Santa Cruz Biotechnology, Santa Cruz, CA), anti-ERK (Santa Cruz), anti-p-AKT (Cell Signaling Technologies, Danvers, MA), anti-AKT (Cell Signaling Technologies, Danvers, MA), anti-Fibronectin (BD Biosciences, San Jose, CA), anti-Fibulin (Abcam, Cambridge, MA), anti-Tubulin (Sigma-Aldrich, St.Louis, MO), anti-Cab45 (OriGene Technologies, Rockville, MD), anti-MMP-2 (Epitomics, Burlingame, CA) and anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA). In addition, HRP-conjugated goat anti-mouse/rabbit IgG was purchased from Invitrogen (Carlsbad, CA).
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4

Quantification of Signaling Pathways

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Cell lysates were extracted with cell lysis buffer (Beyotime, Hangzhou, China) and the protein concentration was quantified using an Enhanced BCA Protein Assay Kit (Beyotime). The primary antibodies used were as follows: anti-MMP2, anti-TIMP1, anti-GAPDH (Epitomics, Hangzhou, China); anti-neogenin (Santa Cruz Biotechnology), anti-netrin 4 (R&D Systems); anti-phosphorylated Stat3, anti-phosphorylated Akt, anti-phosphorylated ERK, anti-phosphorylated p38 (Cell Signaling Technology, Danvers, MA, USA).
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5

Western Blotting for EMT and Apoptosis

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Western blotting analysis was conducted as previously described [41 (link)]. The following primary immunoblotting antibodies were used: anti-GAPDH, anti-E-cadherin, anti-Fibronectin, anti-Vimentin, anti-Snail, anti-Slug, anti-Twist, anti-MMP-9, anti-MMP-2, anti-pro caspase-3, and anti-active caspase-3 (Epitomics, Burlingame, USA), anti-HIF-1α and anti-β-catenin (Proteintech, Chicago, USA), anti-N-cadherin (Cell Signaling Technology, Beverly, MA), and poly (ADP-ribose) polymerase (PARP) (Santa Cruz, CA).
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6

Western Blot Analysis of Proteases

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The assay of western blot was performed as previously described [12 (link)]. The primary antibodies were as follows: anti- CTTN (Cat # ab33333, Abcam), and anti- MT1-MMP (Cat #ab3644, Abcam); anti-MMP2 (Cat #97779, Abcam), anti- MMP9 (Cat # ab38898, Abcam), and anti-GAPDH antibody (Cat #2118, CST). The secondary antibodies were horseradish peroxidase-conjugated anti-rabbit immunoglobulin-G antibody (Cat # ab6721, Abcam).
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7

Histological Analysis of Atherosclerotic Plaques

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The rabbits and mice were euthanized with an overdose of 3% sodium pentobarbital (160 mg/kg i.p.). The rabbit right femoral arteries and mouse hearts containing advanced plaques were prepared as previously described [18 (link)]. Hematoxylin and eosin (H&E) staining was performed for plaque and lumen areas in rabbits as well as for plaque area in mice. Masson’s trichrome staining was performed to determine the collagen content. Immunohistochemical staining was performed to detect macrophages (for rabbits: anti-RAM-11, 1:1200, Dako, Cat#M0633; for mice: anti-CD68, 1:50, Abcam, Cat#ab955), SMCs (for rabbits: anti-α-actin, 1:2000, Sigma, Cat#A2547; for mice: anti-α-actin, 1:500, Abcam, Cat#ab5694), MMP-2 (for rabbits: anti-MMP-2, 1:400, Bioss, Cat#bs-4605R; for mice: anti-MMP-2, 1:50, Abcam, Cat#ab37150), and MMP-9 (for rabbits: anti-MMP-9, 1:400, Bioss, Cat#bs-4593R; for mice: anti-MMP-9, 1:50, Abcam, Cat#ab38898). Images of the stained sections were obtained using an Olympus IX70 microscope (Olympus, Tokyo, Japan). The amount of collagen, macrophages, SMCs, MMP-2, and MMP-9 were measured using computer-assisted color image analysis software (Image-Pro Plus, version 6.0, Media Cybernetics, Inc., Silver Spring, MA, USA).
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8

Western Blot Analysis of Apoptosis and Cell Cycle Markers

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Total protein was extracted using RIPA buffer (Beyotime, Shanghai, China) which contained protease and phosphotase inhibitors cocktails. BCA protein assay kit (Beyotime) was used to determine protein concentrations according to the manufacturer’s instructions. Proteins were separated by SDS-PAGE, transferred to PVDF (Millipore, Burlington, MA, USA) membranes, incubated with primary antibodies overnight at 4°C and then incubated with HRP-conjugated secondary antibody (1:5000 dilution) for 1 h in room temperature. Proteins were visualized by using ECL western blotting detection reagents (Millipore). Immunoreactive bands were quantified using ImageJ (NIH, Bethesda, MD, USA).
The primary antibodies used in this study are purchased from Abcam (Cambridge, UK) as followed: anti-Bax (1:1000), anti-Bcl- 2 (1:1000), anti-caspase 3 (1:1000,), anti-caspase 9 (1:1000), anti- Cyclin D1 (1:1000), anti-PCNA (1:1000), anti-Cox-2 (1:1000), anti-MMP2 (1:1000), anti-MMP9 (1:1000), anti-GTPBP4 (1:1000), and anti-β-actin (1:1000, internal control). All reacts with human proteins.
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9

Protein Expression Analysis by Western Blot

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Cells were lysed in RIPA Lysis Buffer (MA0151, Dalian Meilun Biotechnology Co., Ltd., China) containing a protease inhibitor cocktails (FD1001, Fudebio, Hangzhou, China) on ice. Equal amounts of total protein from different samples were separated by SDS-PAGE gels at 100 V for 1.5 h and transferred onto 0.22 μm polyvinylidene difluoride (PVDF) membranes (Amersham Bioscience, Piscataway, NJ) at 280 mA for 1.5 h. Then, the membranes were blocked with 5% skim milk powder in TBST for 1 h at room temperature and treated with specific primary antibodies overnight at 4 °C. The next day, the membranes were washed with TBST and incubated with an HRP-conjugated secondary antibody (FDM007 and FDR007, Fudebio, Hangzhou, China). Each band was detected using an enhanced chemiluminescence kit (FD8030, Fudebio, Hangzhou, China). Anti-GAPDH, anti-CREB3, anti-Bcl-2, anti-mmp2, anti-Vimentin and anti-E-cadherin antibodies were purchased from Abcam (Cambridge, UK). The anti-c-Jun, anti-cleaved-caspase 3 antibodies were purchased from Cell Signaling Technology (Boston, MA, USA); anti-mmp9 antibody was purchased from ABclonal (Boston, MA, USA); and anti-N-cadherin antibody was purchased from Proteintech (Chicago, USA). Primary Antibody Dilution Buffer was purchased from Dalian Meilun Biotechnology Co., Ltd. (MB9881, Dalian, China).
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10

Cellular Signaling Pathway Profiling

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Celecoxib, cisplatin, 5-fluorouracil (5-FU) and prostaglandin E2 (PGE2) were purchased from Sigma-Aldrich. Each compound was prepared as 1 mM stock solution in dimethylsulfoxide (DMSO) for dilution into various concentrations. The following mouse or rabbit monoclonal primary antibodies were used: anti-E-cadherin (Abcam), anti-MMP-2 (Abcam), anti-Vementin (Abcam), anti-c-MYC (Abcam), anti-Axin-2 (Abcam), anti-Cyclin-D1 (Abcam), anti- β-catenin (Cell Signaling Technology), anti-Survivin (Cell Signaling Technology), anti-SOX-2 (Cell Signaling Technology), anti-p-GSK-3β (Cell Signaling Technology), anti-GAPDH (Beyotime Biotechnology) and anti-Tubulin (Beyotime Biotechnology). HRP conjugated goat anti-mouse or anti-rabbit secondary antibodies were purchased from Beyotime Biotechnology.
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