The mice were killed, and theircollagenase I-treated tendon tissue were fixed with 4% paraformaldehyde, and stained with hematoxylin & eosin to evaluate the change of collagen alignment. Based on our previous study described in detail [22 (link)], a modified semiquantitative 4-point scoring method for each factor was used. Tendinopathy severity, based on the sum of the scores, was graded as 0–3 (0, ≤ 2, 3–4, ≥5 points). After scoring, the tendon tissue were subjected to undergo immunohistochemistry and TUNEL assay. The tendon tissue from normal and overiectomy rats (under static and stretch conditions) were immunohistochemically analyzed. The processed tissuewere snap-frozen and embedded in paraffin. The sections were deparaffinized in xylene, dehydrated in alcohol, treated with proteinase K, washed with H2O2 in PBS, and stained with antibodies against ER-β (Abcam), collagen type I (Abcam), cleaved caspase 3 (Cell Signaling Technology), interleukin-1β (IL)-1β (Cell Signaling), and matrix metalloproteinase-9 (MMP)-9 (Abcam), in combination with the chromogen 3-amino-9-ethylcarbazole (Zymed). The signal intensity was further quantitated using Image J 1.42q (National Institutes of Health) in three randomly chosen fields.
Matrix metalloproteinase 9 mmp 9
Manufactured by Abcam
Sourced in United States
Matrix metalloproteinase-9 (MMP-9) is a zinc-dependent endopeptidase enzyme involved in the breakdown of extracellular matrix and basement membrane components. It plays a role in various physiological and pathological processes.
Automatically generated - may contain errors
Lab products found in correlation
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Gapdh, by Abcam (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
α sma, by Abcam (2 mentions)
Type 1 collagen, by Abcam (1 mentions)
Interleukin 1β il 1β, by Cell Signaling Technology (1 mentions)
3 amino 9 ethylcarbazole, by Zymo Research (1 mentions)
Kn 93, by Merck Group (1 mentions)
U73122, by Merck Group (1 mentions)
P plcγ1 tyr783, by Cell Signaling Technology (1 mentions)
R59949, by Merck Group (1 mentions)
Cleaved parp, by Abcam (1 mentions)
Nf κb, by Cell Signaling Technology (1 mentions)
Cleaved poly adp ribose polymerase parp, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Nfatc1, by Novus Biologicals (1 mentions)
Recombinant macrophage colony stimulating factor m csf, by R&D Systems (1 mentions)
8 protocols using matrix metalloproteinase 9 mmp 9
1
Histological Evaluation of Tendinopathy
2
Signaling Pathway Protein Analysis
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Antibodies against PLCγ1, p-PLCγ1 (Tyr783), PKCδ, CaMK IIβ, Akt, p-Akt (S473), ERK, p-ERK (Thr202/Tyr204), mTOR, p-mTOR (Ser2481), p-S6 (Ser235/236), and NF-κB were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). Cleaved-PARP, Matrix metalloproteinase-9 (MMP9), and GAPDH were purchased from Abcam (Cambridge, MA, USA). Inhibitors (U73122, KN93, and R59949) were obtained from Sigma-Aldrich in China (Shanghai, China). Other reagents were of the highest grade commercially available.
3
Western Blot Analysis of Protein Expression
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Western blot assays were performed as described previously.12 (link) In brief, extracted proteins from tissues and cell lines were separated
by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then
transferred to polyvinylidene fluoride membranes (Bio-Rad Laboratories, USA).
After blocking, the membranes were incubated with a primary antibody overnight
at 4°C. Then, the blotted membranes were incubated with a horseradish
peroxidase-conjugated secondary antibody (1:2000) for 2 h at room temperature.
Labeled proteins were detected using enhanced chemiluminescence following the
manufacturer’s protocol. β-Actin (1:1000, Cell Signaling, USA) was used as an
internal control. Antibodies against the following proteins were used:
E-cadherin (1:1000, Cell Signaling), matrix metalloproteinase-9 (MMP-9; 1:1000,
Abcam, USA), vimentin (1:1000, Cell Signaling), cleaved caspase-3 (1:1000, Cell
Signaling) and cleaved poly (ADP-ribose) polymerase (PARP; 1:1000, Cell
Signaling).
by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then
transferred to polyvinylidene fluoride membranes (Bio-Rad Laboratories, USA).
After blocking, the membranes were incubated with a primary antibody overnight
at 4°C. Then, the blotted membranes were incubated with a horseradish
peroxidase-conjugated secondary antibody (1:2000) for 2 h at room temperature.
Labeled proteins were detected using enhanced chemiluminescence following the
manufacturer’s protocol. β-Actin (1:1000, Cell Signaling, USA) was used as an
internal control. Antibodies against the following proteins were used:
E-cadherin (1:1000, Cell Signaling), matrix metalloproteinase-9 (MMP-9; 1:1000,
Abcam, USA), vimentin (1:1000, Cell Signaling), cleaved caspase-3 (1:1000, Cell
Signaling) and cleaved poly (ADP-ribose) polymerase (PARP; 1:1000, Cell
Signaling).
4
Osteoclastogenesis Signaling Pathway
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Recombinant macrophage colony-stimulating factor (M-CSF) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA) and recombinant RANKL was purchased from Abcam (Cambridge, MA, USA). For western blotting analysis, the following antibodies were used: c-Jun N-terminal kinase (JNK) (R &D Systems Inc.), Matrix metalloproteinase 9 (Mmp-9) (Abcam), c-Fos, p-p42/44, p42/44, p-AKT, and AKT (Cell Signalling Technology, Massachusetts, MA, United States); Gapdh, p38, and IκBα, HRP anti-mouse, and HRP anti-rabbit (Santa Cruz Biotechnology, San Diego, CA, USA); p-p38 and p-JNK (New England Biolabs, Ipswich, MA, USA); NFATc1 (Novus Biologicals, Littleton, CO, USA).
5
Western Blot Analysis of Proteins
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Total protein was extracted from rat pulmonary arteries or HPASMCs and its concentration was measured by BCA kit (Elabsience, China). Primary antibodies we used were as follows: GPS2 (1:1000), ERK1/2 (1:3500), p-ERK1/2 (Thr202/Tyr204) (1:3500) (Wuhan Sanying, China), Raf1 (1:1000), p-Raf1 (ser259) (1:1000) (CST, USA), proliferating cell nuclear antigen (PCNA) (1:3000), matrix metalloproteinase 9 (MMP9) (1:1000), TP53 (1:1000) (Abcam, UK), and the relevant secondary antibodies. Finally, ECL luminescent solution was applied to develop the color; a fully automated chemiluminescence gel-imaging analyzing system (Beijing Sage Science and Technology Co., Ltd.) was used for exposure and photography; and Image J software was utilized for analysis. Quantitative results are expressed as gray value ratios of target proteins to GAPDH or β-Actin, except for the signaling pathway proteins, such as “Active-Ras/Ras, p-Raf/Raf, and p-ERK/ERK” which were presented as a ratio of each phosphorylated protein to their respective total proteins.
6
Western Blot Analysis of Tissue Proteins
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Tissue proteins were extracted by RIPA lysis buffer (Cell Signaling Technology, USA) containing a protease inhibitor cocktail (KeyGEN BioTECH, China). The protein concentration was determined by the BCA protein assay (Beyotime, China). After that, the proteins were isolated by SDS–PAGE and then passed on to a PVDF membrane (Millipore, USA). The membrane was blocked with 5% bovine serum albumin at room temperature for 1 h and then incubated with primary antibodies against collagen I (Abcam, USA), matrix metalloproteinase 9 (MMP9) (Abcam, USA), IGFBP3 (Abcam, USA), B-cell lymphoma-2 (Bcl-2) (Abcam, USA), Bcl-2-associated X protein (Bax) (Abcam, USA), and GAPDH (Abcam, USA). The membrane was washed and soaked with appropriate secondary antibodies at room temperature for 1 h. The protein bands were visualized using enhanced chemiluminescence (ECL) reagents (Millipore, USA) and imaged with an imaging system (Tanon, China). The GAPDH signal was used as a loading control.
7
Aortic Protein Extraction and Western Blot Analysis
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Briefly, protein was extracted from the aorta using radioimmunoprecipitation assay (RIPA) buffer (Beyotime, China) and quantified using a BCA Protein Kit (CWBio, China). Proteins were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked with 1% bovine serum albumin (BSA) for 1 h, and then incubated with primary antibodies against p-AKT (Cell Signaling Technology, USA), AKT (Cell Signaling Technology, USA), p-mTOR (Cell Signaling Technology, USA), mTOR (Cell Signaling Technology, USA), p62 (Cell Signaling Technology, USA), LC3 (Sigma-Aldrich, USA), SM22α (Abcam, UK), α-SMA (Abcam, UK), myocardin (Abcam, UK), OPN (Abcam, UK), matrix metalloproteinase-2 (MMP-2; Novus, USA), matrix metalloproteinase-9 (MMP-9; Abcam, UK), p65 (Cell Signaling Technology, USA), TNF-α (Abcam, UK), and β-actin (Abcam, UK), overnight at 4°C. Next, membranes were washed three times with tris buffered saline tween (TBST) and incubated for 1 h with secondary antibodies (Abcam, UK). Proteins were detected using enhanced chemiluminescent reagents (Thermo Fisher Scientific, USA) and quantified using ImageJ software (NIH, USA).
8
Whole-Cell Lysate and Nuclear Extract Preparation for Western Blot Analysis
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Whole-cell lysates were prepared in RIPA buffer and nuclear extracts were prepared as previously described. 21 Aliquots of protein were then subjected to western blot analysis. Ponceau S (Sigma) staining was used to verify equal loading and transfer. Primary antibodies used were: iNOS (BD Biosciences), hypoxia-inducible factor 1(HIF-1) α (Novus Biologicals), αSMA, matrix metalloproteinase-9 (MMP-9; Abcam), and histone 2AX (H2AX) phospho-specific (Ser139; Cell Signaling Technology). Secondary antibodies were obtained from Jackson ImmunoResearch (West Grove, PA).
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