ChIP assay was conducted using an EZ ChIP™ Chromatin Immunoprecipitation Kit (Millipore). In summary, sonication was used to break the crosslinked chromatin into 200‐1000 bp fragments. Anti‐SOX18 (1:200 dilution, Cell Signaling Technology) and anti‐MMP7 (1:1000 dilution, Abcam) antibodies were then used to immunoprecipitate the chromatin.
Anti mmp7
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Anti-MMP7 is a laboratory reagent used in research applications. It is an antibody that specifically binds to and detects the MMP7 (Matrix Metalloproteinase-7) protein. MMP7 is an enzyme involved in the breakdown of extracellular matrix proteins. This antibody can be used to study the expression and function of MMP7 in various biological systems.
Automatically generated - may contain errors
Lab products found in correlation
Anti mmp2, by Abcam (4 mentions)
Anti sox18, by Cell Signaling Technology (3 mentions)
Anti mmp9, by Abcam (3 mentions)
Chemiluminescence, by Merck Group (2 mentions)
Anti c myc, by Santa Cruz Biotechnology (2 mentions)
Anti ctnnb1, by Santa Cruz Biotechnology (2 mentions)
Anti ccnd1, by Santa Cruz Biotechnology (2 mentions)
Anti mmp 3, by Abcam (2 mentions)
Anti slc12a5, by Abcam (2 mentions)
Anti vimentin, by Abcam (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Ez chip chromatin immunoprecipitation kit, by Merck Group (1 mentions)
Anti mmp14, by Abcam (1 mentions)
Anti mmp12, by Abcam (1 mentions)
Anti mmp1, by Abcam (1 mentions)
Anti mmp8, by Abcam (1 mentions)
Anti mmp9, by Thermo Fisher Scientific (1 mentions)
Anti timp 2, by Bioss Antibodies (1 mentions)
Anti collagen, by Abcam (1 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)
12 protocols using anti mmp7
1
ChIP Assay for SOX18 and MMP7
2
Western Blot Analysis of Protein Markers
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WB was performed as previously described 17 (link). After incubation with the secondary antibody, the proteins were detected by chemiluminescence (Millipore Germany). The primary antibodies used in this study were anti-LHX6 (1:1000; Santa Cruz Biotechnology), anti-CTNNB1 (1:700; Santa Cruz Biotechnology), anti-MMP7 (1:1000; Abcam), anti-c-Myc (1:1000; Santa Cruz Biotechnology), anti-CCND1 (1:1000; Santa Cruz Biotechnology) and anti-GAPDH (1:2000; Beyotime China).
3
Quantification of Matrix Metalloproteinases in Cancer
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For preparation of protein extracts, 12 pairs of cancer and adjacent normal tissues were crushed with a mortar under ice cold conditions and lysed with RIPA lysis buffer together with protease inhibitors. Cells were collected and lysed with RIPA lysis buffer together with protease inhibitors. After centrifugation at 12,000 rpm at 4°C for 20 min, supernatants were collected and protein concentration was determined using the Pierce™ BCA protein assay (Thermo, Waltham, MA, USA). Proteins were separated by electrophoresis on a 10% SDS-polyacrylamide gel, electroblotted onto a PVDF membrane, and blocked by 5% nonfat dry milk for 1 h. Membranes were then washed in TBST three times for 5 min and then incubated with anti-MMP1 (Abcam, Cambridge, MA, USA), anti-MMP2 (Abcam), anti-MMP3 (Abcam, USA), anti-MMP7 (Abcam, USA), anti-MMP8 (Abcam, USA), anti-MMP9 (Invitrogen, Waltham, MA, USA), anti-MMP11 (Bioss, Beijing, China), anti-MMP12 (Abcam, USA), anti-MMP14 (Abcam, USA), anti-MMP17 (Abcam, USA), anti-MMP19 (Bioss, China), anti-MMP28 (Abcam, USA), anti-Collagen (Abcam), anti-TIMP2 (Bioss, China), or anti-GAPDH (Abcam). Subsequently, the membranes were washed with PBST and incubated for 1 h with goat anti-rabbit IgG (Abcam). Finally, membranes were washed three times and immunoreactivity was determined by using a Chemi DOC™ XRS+ system (BioRad Laboratories, Hercules, CA, USA).
4
Histological Analysis of Intestinal Tumors
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Tissues were fixed in 10% neutral buffered formaldehyde for 4–12 h, transferred into 70% ethanol and processed by standard techniques. Sections (5 μm) were stained with hematoxylin and eosin. For immunostaining, antigens were retrieved by 10-min boiling in 10 mM citrate (pH 6.0). Blinded histological inflammatory scores were performed by a validated scoring system.40 (link) Histological assessment of intestinal tumors in the rodent was performed using the recommended standards.41 (link) Immunostaining, including BrdU staining, was performed as previously described.15 (link), 42 (link) The slides were stained with anti-BrdU, anti-Bmi1, anti-phospho-c-Myc, anti-cyclin D1, anti-MMP-7 (Abcam, Cambridge, MA, USA), anti-phospho-β-catenin (Ser552), anti-β-catenin (BD, Transduction Laboratories, Lexington, KY, USA).
5
Immunohistochemical Staining Evaluation Protocol
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After staining with primary antibodies, a Dako Real Envision Kit (Dako) was used to visualize protein expression, according to the manufacturer's instructions. Two independent experienced pathologists scored the staining intensity manually. To evaluate IHC staining, previously published scoring criteria for SLC12A5,13 SOX18,17 or MMP713 were used. Primary antibodies comprised: anti‐SLC12A5 (1:100 dilution, Abcam), anti‐SOX18 (1:200 dilution, Cell Signaling Technology), and anti‐MMP7 (1:1000 dilution, Abcam).
6
Comprehensive Protein Expression Analysis
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Protein was extracted from the cells and quantified by a Total Protein Extraction Kit (Keygen BioTech, Nanjing, China). Western blotting was performed according to the manufacturer’s protocol. Anti-TPPP, anti-YY1 (#ab109228), anti-E-cadherin (#ab40772), anti-vimentin (#ab92547), anti-MMP3 (#ab52915), anti-MMP7 (#ab205525), anti-VEGF (#ab32152), anti-p38 (#ab170099), anti-MAPK (#ab205926), anti-p38 MAPK (phosphor, Thr180/Tyr182, #4511S), anti-PI3K (#4255S), anti-PI3K (phosphor, Ser249, #13857S), anti-AKT (#2920S), anti-AKT (phosphor, Thr308, #13038S), anti-β-actin (#3700S) and anti-YY1 (#ab12132) antibodies for ChIP were obtained from Abcam (Cambridge, MA) or Cell Signaling Technology (Danvers, MA, USA). β-Actin was used as an endogenous reference. Each blot was independently repeated three times.
7
Western Blot Analysis of Bladder Cancer Cells
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The transfected J82 and T24 cells were lysed using protein inhibitor modified RIPA lysate (Thermo Fisher) on ice, and the total cell protein was extracted. After accurate loading, electrophoresis separation was performed on a 10% SDS-PAGE gel, and then transferred to a superior PVDF membrane. The PVDF membrane was blocked with skim milk (BD) for 2 h. The following primary antibodies were added and incubated overnight at 4 °C, anti-SHMT2(1:1,000, ab224428; Abcam, Cambridge, UK), anti-Tubulin(1:10,000, ab176560; Abcam, Cambridge, UK), anti-MMP7(1:1,000, ab205525; Abcam, Cambridge, UK), anti-Integrin β-4(1:1,000, ab182120; Abcam, Cambridge, UK), anti-Laminin β-4(1:500, Sc-130540; Santa Cruz, CA, USA), anti-SHMT1(1:800, Cat.14149–1-AP; Proteintech, Wuhan, China), anti-E-cadherin (1:2,000, Cat.60335–1-Ig; Proteintech, Wuhan, China), anti-N-cadherin (1:2,000, Cat.66219–1-Ig; Proteintech, Wuhan, China), anti-MMP2 (1:2,000, Cat.66366–1-Ig; Proteintech, Wuhan, China), anti-GAPDH (1:10,000, Cat.60004–1-Ig; Proteintech, Wuhan, China). The membrane was washed the next day and secondary antibodies were added and incubated for 30 min. After washing, the membrane was treated with ECL chemiluminescence reagent for 1 min and finally imaged using a fluorescence imaging system. The gray value of protein bands was analyzed using Image J software and the experimental data were recorded.
8
Epithelial-Mesenchymal Transition Protein Assay
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Quercetin (Sigma-Aldrich Co., St Louis, MO, USA) was stored at −80°C until dilution in DMSO before use. Fetal bovine serum (FBS), trypsin, RPMI-1640, and DMEM were purchased from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). Anti-N-cadherin, anti-Vimentin, anti-Twist, anti-Slug, anti-MMP-2, and anti-MMP-7 antibodies were purchased from Abcam (Cambridge, UK). Anti-E-cadherin, anti-Zeb1, anti-Snail, anti-STAT3, and anti-phospho-STAT3 antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Polymerase chain reaction (PCR) primers were purchased from Synbio Tech (Jiangsu, People’s Republic of China). Power SYBR Green PCR Master Mix was purchased from Applied Biosystems (Foster City, CA, USA) and a Revert Aid First Strand cDNA Synthesis Kit was purchased from Thermo Fisher Scientific.
9
Western Blot Analysis of Signaling Proteins
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WB was performed as previously described [9 (link)]. After incubation with the secondary antibody, the proteins were detected by chemiluminescence (Millipore Germany). Primary antibodies used in this study were anti-ALX4 (1:1000; Santa Cruz Biotechnology), anti-CTNNB1 (1:700; Santa Cruz Biotechnology), anti-p-CTNNB1 (1800; Santa Cruz Biotechnology), anti-GSK3β (1:800; Santa Cruz Biotechnology), anti-c-Myc (1:1000; Santa Cruz Biotechnology), anti-CCND1 (1:1000; Santa Cruz Biotechnology), anti-MMP7 (1:1000; Abcam) and anti-GAPDH (1:2000; Beyotime China). The siRNA targeting human GSK3β was purchased from Riobio (stQ0004712–1).
10
Protein Expression Analysis by Western Blot
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Cells were lysed using lysis buffer to obtain total proteins, which were separated using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). PVDF membranes were blocked for 1 h, then the membranes were incubated with monoclonal antibodies overnight at 4°C. Secondary antibodies were then reacted with the membranes for 1 h at room temperature. Enhanced chemiluminescence (ECL kit, Santa Cruz Biotechnology Inc) was used to visualize the immunoreactive protein bands and the band intensities were analyzed using the Quantity One system (Bio‐Rad). Primary antibodies comprised: anti‐SLC12A5 (1:1000 dilution, Abcam), anti‐SOX18 (1:1000 dilution, Cell Signaling Technology), anti‐MMP7 (1:1000 dilution, Abcam), anti‐MMP2 (1:800 dilution, Abcam), anti‐MMP9 (1:1000 dilution, Abcam), and anti‐GAPDH (1:5000 dilution, Cell Signaling Technology). For western blotting experiments, CHX was used at 100 μg/mL for 24 h (Sigma‐Aldrich).
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