Cell lysates were harvested from A498 and 786-O cells with or without treatment of different dose of Rhein by RIPA buffer (Beyotime). Protein lysates were dissolved in 2× loading buffer and boiled for 15 min at 100°C. After that protein lysates were resolved on 11% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred to nitrocellulose membrane. Resultant blots were blocked with 5% skim milk and reacted with properly diluted primary antibodies against glyceraldehyde 3-phosphate dehydrogenase (Santa Cruz), phospho-extracellular signal-regulated kinase (Santa Cruz), total extracellular signal-regulated kinase (Santa Cruz), p-AKT (Cell Signaling Technology), phospho-c-Jun N-terminal kinase (Cell Signaling Technology), matrix metalloproteinase 9 (MMP9) (Cell Signaling Technology) and CCND1 (Cell Signaling Technology) for 1 h at room temperature. Immune complexes were detected by horseradish peroxidase-conjugated secondary antibodies, followed by enhanced chemiluminescence reaction (Amersham Pharmacia Biotech).
Matrix metalloproteinase 9 mmp9
Manufactured by Cell Signaling Technology
Sourced in United States, China
Matrix metalloproteinase 9 (MMP9) is an enzyme that belongs to the matrix metalloproteinase family. Its core function is the breakdown of extracellular matrix proteins.
Automatically generated - may contain errors
Lab products found in correlation
P akt, by Cell Signaling Technology (2 mentions)
Mmp 2, by Cell Signaling Technology (2 mentions)
β actin, by Merck Group (2 mentions)
Ccnd1, by Cell Signaling Technology (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase, by Santa Cruz Biotechnology (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Abcg2, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Abcam (1 mentions)
Cox 4, by Cell Signaling Technology (1 mentions)
Notch1, by Cell Signaling Technology (1 mentions)
Nicd1, by Cell Signaling Technology (1 mentions)
2 2 2 trichloroethanol, by Merck Group (1 mentions)
Phospho akt, by Cell Signaling Technology (1 mentions)
Phospho histone h2ax γh2ax, by Cell Signaling Technology (1 mentions)
Claudin 1, by Cell Signaling Technology (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
8 protocols using matrix metalloproteinase 9 mmp9
1
Rhein Modulates Signaling Pathways in Renal Cancer Cells
2
Immunoblotting Analysis of Apoptosis Proteins
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Immunoblotting was performed to detect apoptosis–associated molecules. After BB treatment, the cells were lysed with RIPA buffer (Pierce, USA), and protein concentration was quantified by means of the Bradford protein assay (Bio-Rad, USA). A total cell lysate was prepared as described elsewhere [18 (link)]. Protein samples were resolved by SDS-PAGE and transferred onto a PVDF membrane (Bio-Rad). The membranes were probed with antibodies against cleaved caspase 3, cleaved PARP, p38, phosphorylated p38, Matrix Metalloproteinase-9(MMP-9) and β-actin (Cell Signaling Technology, USA). Signals were detected with secondary antibodies and Pierce ECL-Plus chemiluminescence (Thermo Scientific, USA).
3
Protein Expression Profiling in Leukemic Cells
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After transfection with miR-98 mimic for 48 h, the cells were washed, harvested and lysed to extract the total protein. The concentration was estimated by bicinchoninic acid assay, and 50 µg protein was resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. GAPDH (Cell Signaling Technology Inc, Boston, MA, USA) was used as a loading control to assess the differences in the expression of E2F1 and ABCG2 in K562 and K562/A02. The Western blot membrane was probed with E2F1 (Abcam Inc, Wuhan, People’s Republic of China), ABCG2 (Cell Signaling Technology), Bax (Abcam), p21 (Abcam), matrix metalloproteinase 9 (MMP9; Cell Signaling Technology) primary antibodies at 1:1,000. Subsequently, goat anti-rabbit and goat anti-rat secondary antibodies were added. ImageJ (National Institutes of Health, Bethesda, MD, USA) was used to analyze the images.
4
Immunoblotting Assay Protocol
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Immunoblotting was performed as described (Ehrlich et al, 2015 (link)). The following primary antibodies were used: AKT (9272, Cell Signaling), AKT phospho (Ser 473; 4051, Cell Signaling), β-actin (MAB1501, Millipore), β-tubulin (2146, Cell Signaling), Bim (2819, Cell Signaling), Cdk5 (AHZ0492, Life Technologies), COX IV (4844, Cell Signaling), CREB (9104, Cell Signaling), ERK (9102, Cell Signaling), ERK phospho (9106, Cell Signaling), Foxo1 (2880, Cell Signaling), GAPDH (sc-69778, Santa Cruz), HIF1α (610958, BD Biosciences), phospho-histone H2aX (γH2aX, 2577, Cell Signaling), matrix metalloproteinase-9 (MMP-9; 3852 Cell Signaling), MMP-2 (4022 Cell Signaling), NICD-1 (4147, Cell Signaling), NICD-4 (sc-5594, Santa Cruz), Notch 1 (3608, Cell Signaling), Retinoblastoma protein phospho (Ser807/811; 8516, Cell Signaling), Retinoblastoma protein (554136, BD Biosciences), Stat3 phospho (Ser 727; 9134, Cell Signaling), Stat3 (9132, Cell Signaling). Anti-vimentin, -snail, -β-catenin, -claudin-1, N-cadherin were from Cell Signaling (EMT Sampler Kit, 9782), c-Myc (sc-788, Santa Cruz). Loading control by stain-free gels was performed by adding 0.5% TCE (2,2,2-Trichloroethanol, Sigma-Aldrich) to the PAGE gels according to the TGX Stain-Free Gels system (BioRad, Hercules, CA, USA).
5
Western Blot Analysis of Apoptosis Markers
6
Protein Expression Analysis by Western Blot
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Cells were harvested and lysed in RIPA buffer with a protease inhibitor cocktail (1183170001, Roche, Hvidovre, Denmark) and phosphatase inhibitor cocktail (04906837001, Roche, New York, NY, USA). Total protein lysates were separated by 10% SDS-PAGE and transferred to Nitrocellulose membranes (66485, Pall Corporation, Port Washington, NY, USA). Membranes were then incubated with primary antibodies at 4 °C overnight. Subsequently, membranes were incubated with secondary antibodies at room temperature. Western blot analyses were carried out using the following antibodies: YKT6 (cat# sc-365732, Santa Cruz Biotechnology), matrix metalloproteinase 9 (MMP-9) (cat# 13667, Cell Signaling Technology, Danvers, MA, USA), and β-actin (cat# A5316, Sigma-Aldrich, St. Louis, MO, USA). Signals were visualized by enhanced chemiluminescence assays (Bio-Rad, Hercules, CA, USA).
7
SSA Regulates CXCR4/CXCR7 and AKT/mTOR Pathways
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After SUM149 and MDA-MB-231 cells were treated with SSA (0, 2.5, 5, or 10 μM) for 24 h, total cell protein lysates were extracted using RIPA lysis buffer that contained a protease inhibitor cocktail and a phosphatase inhibitor cocktail. Protein lysates (40 μg), determined by BCA analysis (Beyotime, Shanghai, China), were then subjected to Western blotting analysis. The primary antibodies used in analyses were as follows: CXCR4 (1:1,000, Novus, USA), CXCR7 (1:1,000, Proteintech, China), AKT (1:500, Cell Signaling Technology, USA), p-AKT (1:2,000, Cell Signaling Technology, USA), mammalian target of rapamycin (mTOR, 1:1,000, Proteintech, China), matrix metalloproteinase-9 (MMP9, 1:500, Cell Signaling Technology, USA), MMP2 (1:500, Cell Signaling Technology, USA), and caspase 3 (1:1,000, Proteintech, China). The second antibody was a goat anti-rabbit secondary antibody coupled to horseradish peroxidase (1:20,000, Proteintech, China).
To further investigate the effect of SSA on the expression of these proteins in vivo, lungs from each group were collected and subjected to Western blotting as described above.
To further investigate the effect of SSA on the expression of these proteins in vivo, lungs from each group were collected and subjected to Western blotting as described above.
8
Protein Expression Analysis by Western Blot
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Protein extraction and western blot analysis were performed as per the protocols described earlier. 15 Briefly, MUT-MCF-7 and hGH-MCF-7 cells were lysed with hot sample buffer containing 1% sodium dodecyl sulfate (SDS) and then centrifuged for 15 min at room temperature. Protein extracts were separated by 10% SDSpolyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride (PVDF; Hybond C-Extra; Amersham Pharmacia, Aylesbury, UK). After blocking, membranes were incubated with primary anti-mouse monoclonal antibodies (NF-κB (1:1000), E-cadherin (1:1000), P-cadherin (1:1000), and matrix metalloproteinase-9 (MMP-9; 1:1000) obtained from Cell Signaling Technology, Beverly, MA, USA)) for 1 h. The membranes were then washed and incubated with horseradish peroxidase-conjugated goat anti-mouse secondary antibody (1:10,000; Upstate Biotechnology, Lake Placid, NY, USA).
Blots were stripped and reprobed using anti-actin antibody (Sigma Aldrich, St. Louis, MO, USA). Protein bands were detected by the Phototype horseradish peroxidase western blot detection system (Cell Signaling Technology).
Blots were stripped and reprobed using anti-actin antibody (Sigma Aldrich, St. Louis, MO, USA). Protein bands were detected by the Phototype horseradish peroxidase western blot detection system (Cell Signaling Technology).
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