Aortic tissues were homogenized and subjected to centrifugation at 4 °C for 10 min (12,000 rpm), the supernatant was extracted. Protein mixed with loading buffer were heated at 100 °C for 5 min. Extractions were then separated with a 10 % SDS-PAGE, electrotransferred to PVDF membranes and blocked with 5 % bovine serum albumin. The membranes were incubated with primary antibodies against TH (1:800, Boster, China), ChAT (1:800, Boster, China), MMP2 (1:1000, Cell Signaling Technology, USA), matrix metalloproteinase 9 (1:1000, Cell Signaling Technology, USA) overnight at 4 °C and followed by fluorescent tags secondary antibodies (1:10000, LI-COR, USA) for 1 h at room temperature. Detection of fluorescence was performed by Odyssey Imaging system (LI-COR, USA). For all the target proteins, GAPDH (1:1000, Cell Signaling Technology, USA) was chosen as an internal control.
Matrix metalloproteinase 9
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
Matrix metalloproteinase 9 (MMP-9) is a laboratory product that functions as an enzyme involved in the breakdown of extracellular matrix proteins. It plays a role in various physiological and pathological processes.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Cell Signaling Technology (2 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Mmp 2, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Hypoxia inducible factor 1α hif 1α, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Abcam (1 mentions)
Enhanced chemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
E cadherin, by Abcam (1 mentions)
Gapdh, by Abcam (1 mentions)
Bcl 2, by Abcam (1 mentions)
2 protocols using matrix metalloproteinase 9
1
Aortic Tissue Protein Analysis
2
Western Blot Analysis of Protein Expression
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Protein was extracted from tissue specimens and cultured cells using RIPA lysis buffer (Beyotime, Shanghai, China) and the concentration was determined using a BCA Protein Assay Kit (Beyotime). Equal amounts of protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene-fluoride membranes (Millipore, Bedford, MA). After blocking with 5% non-fat milk in Tris-buffered saline and Tween 20 for 1 h at room temperature, the membranes were incubated with primary antibodies against Nrf2, HO-1, proliferating cell nuclear antigen (PCNA), cyclin D1, Bcl-2, Bax (Abcam, Cambridge, UK), E-cadherin, vimentin, matrix metalloproteinase-9 (MMP-9), hypoxia-inducible factor 1α (HIF-1α), vascular endothelial growth factor (VEGF), and GAPDH (Cell Signaling Technology Beverly, MA) at 4°C overnight, followed by incubation with horseradish peroxidase (HRP)conjugated secondary antibodies (Abcam) at 37°C for 1 h. GAPDH was used as the loading control. Protein bands were analyzed using an enhanced chemiluminescence kit (Pierce Biotechnology, Rockford, IL) with an imaging system (Bio-Rad Laboratories, Hercules, CA).
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