VSMCs were lysed in RIPA buffer. Protein concentrations were quantified with protein assay reagent (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were loaded and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes (Bio-Rad). The membranes were incubated overnight with primary antibodies against HIF-1α (1:1000, Abcam), calpastatin, TGF-β1 (1:2000, Abcam), calpain-1, calpain-2, a-II spectrin (1:3000, Abcam), MMP2, TIMP2 (1:400, Boster), MT1MMP (1:200, Boster), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:1000, Cell Signaling Technology). At room temperature, membranes were incubated with corresponding secondary antibodies for 1 hour. Quantitative analysis was performed with Image J software (NIH). All samples were run in triplicate and averaged.
Timp2
Manufactured by Boster Bio
TIMP2 is a lab equipment product that functions as a tissue inhibitor of metalloproteinases. It is a protein that regulates the activity of matrix metalloproteinases, which are enzymes involved in the breakdown and remodeling of extracellular matrix.
Automatically generated - may contain errors
Lab products found in correlation
Calpastatin, by Abcam (2 mentions)
Calpain 1, by Abcam (2 mentions)
Mt1mmp, by Boster Bio (2 mentions)
Calpain 2, by Abcam (1 mentions)
Tgf β1, by Abcam (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Hif 1α, by Abcam (1 mentions)
Protein assay reagent, by Bio-Rad (1 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (1 mentions)
Mmp 8, by Boster Bio (1 mentions)
Tgf β1, by Boster Bio (1 mentions)
Mmp 7, by Boster Bio (1 mentions)
Timp 1, by Boster Bio (1 mentions)
Collagen 1, by Boster Bio (1 mentions)
3 protocols using timp2
1
Protein Expression Analysis in VSMCs
2
Protein Expression Analysis in Rat Kidney
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A frozen kidney tissue of rats in each group was taken to extract proteins and estimate the protein by bicinchoninic acid assay (BCA) colorimetric method. The protein was denatured and separated by SDS-PAGE electrophoretic technique and transferred to a polyvinylidene difluoride (PVDF) membrane. After formulating with 5% bovine serum albumin (BSA) for 2 h, the blocked membranes were incubated with ERK1/2 (cell signaling), TGF-β1, MMP-2, MMP-7, MMP-8, MMP-11, MMP-14, TIMP1, and TIMP2 (Boster Biological Technology, Ltd., Wuhan, PR China) antibody, and then washed with tris buffered saline with Tween (TBST), and detected with a secondary antibodies (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD), and finally these membranes were subjected to chemiluminescence detection assay.
3
Carotid Artery Histological Analysis
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After perfusion with saline, carotid arteries were fixed with 4% paraformaldehyde and embedded in paraffin. Thin sections (4–5 μm) were stained with haematoxylin & eosin (HE). The areas of intima and media of the carotid artery were measured using Image-Pro Plus software. Cell proliferation was assessed by immunohistochemical staining of proliferation cell nuclear antigen (PCNA) with the corresponding antibody (1:200, Abcam). Protein expression was evaluated by immunohistochemical staining using antibodies against calpastatin, calpain-1, calpain-2 (1:100, Abcam), MMP2, MT1MMP, TIMP2, and collagen I (1:100, Boster).
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