Total cell lysate and ECM were isolated and resolved by SDS-PAGE in 4% to 20% gels as previously described.17 (link) Resolved gels were transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA) and probed with the following primary antibodies: ACTN (1:750; Santa Cruz Biotechnology, Dallas, TX, USA), BEST1 (1:500; Millipore, Billerica, MA, USA), COL4 (1:1000; Abcam, Cambridge, MA, USA), CRALBP (1:10000),25 (link) EZR (1:1000, Cell Signaling Technology, Danvers, MA, USA), LAM (1:1000; Abcam), OCLN (1:1000; Thermo Fisher Scientific), RPE65 (1:500; Millipore), RHO (1:500; Millipore), and TIMP3 (1:250; Abcam). Secondary antibodies were host-specific near-infrared (1:12,500) (LiCor, Lincoln, NE, USA) or horseradish peroxidase conjugated (1:10,000) (Jackson ImmunoResearch, West Grove, PA, USA) and signals were detected on the LiCor Odyssey or the Azure C500 (Azure Biosystems, Dublin, CA, USA) imaging systems. After image acquisition, Western blot data were analyzed quantitatively using LiCor Odyssey 3.0 and/or Image Studio Lite version 5.2 (LiCor) and Microsoft Excel (Redmond, WA, USA).
Timp3
Manufactured by Abcam
Sourced in United States, United Kingdom
TIMP3 is a protein that acts as an inhibitor of matrix metalloproteinases (MMPs), which are enzymes involved in the breakdown of the extracellular matrix. TIMP3 plays a role in the regulation of tissue remodeling and degradation.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510 meta confocal microscope, by Zeiss (3 mentions)
Mmp 9, by Abcam (2 mentions)
Timp 1, by Abcam (2 mentions)
Timp 2, by Abcam (2 mentions)
Mcp 1, by Abcam (2 mentions)
Licor odyssey 3, by LI COR (1 mentions)
Image studio lite version 5, by LI COR (1 mentions)
Azure c500, by Azure Biosystems (1 mentions)
Rpe65, by Merck Group (1 mentions)
Horseradish peroxidase conjugated, by Jackson ImmunoResearch (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
P c raf, by Cell Signaling Technology (1 mentions)
P akt thr308, by Cell Signaling Technology (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
P akt ser473, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Bioss Antibodies (1 mentions)
Hrp labeled goat anti rabbit or goat anti mouse secondary antibodies, by Cell Signaling Technology (1 mentions)
Caspase 9, by Cell Signaling Technology (1 mentions)
Tsg101, by Abcam (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
16 protocols using timp3
1
Quantitative Western Blot Analysis
2
Comprehensive Protein Analysis Protocol
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Total proteins were extracted from cells using Whole Cell Lysis Assay (KeyGEN BioTECH, China) on ice and quantified by bicinchoninic acid protein assay kit (Beyotime, Shanghai, China). The western blot was performed according to standard procedures. The following primary antibodies were applied: MIF (Novus Biologicals, USA), TIMP3, PI3K, CD81, TSG101, ALIX (Abcam, USA), caspase-3, caspase-9, P-c-Raf, AKT, P-Akt (Ser473), P-Akt (Thr308), β-actin (Cell Signaling Technology, USA), BAX (Bioss ANTIBODIES, USA), P21 (Proteintech, USA), CDK2 (ImmunoWay, USA), and Cyclin H (ZenBio Science, China). The antibody information is listed in Table S2 . After primary antibody incubation, the membranes were incubated with HRP-labeled goat anti-rabbit or goat anti-mouse secondary antibodies (Cell Signaling Technology, USA). The bands were captured by chemiluminescence (Biosciences, Foster City, CA, USA) through ECL detection system. Finally, the protein expression was analyzed by the software of ImageJ.
3
Western Blot Analysis of Cell Signaling Proteins
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The lysates were resolved by 12% SDS-PAGE and then transferred to PVDF membranes. Primary antibodies against the following were used at 4 degree overnight: MMP-2 (Abcam, CA, USA); TIMP3 (Abcam, CA, USA); Cyclin D1 (Cellular Signaling Technology, MA, USA); Cyclin E1 (Cellular Signaling Technology, MA, USA); CDK4 (Cellular Signaling Technology, MA, USA); p21(Cellular Signaling Technology, MA, USA); Caspase-8 (Cellular Signaling Technology, MA, USA); Caspase-3 (Cellular Signaling Technology, MA, USA); cleaved PARP (Cellular Signaling Technology, MA, USA); Bcl-2 (Cellular Signaling Technology, MA, USA); Mcl-1 (Cellular Signaling Technology, MA, USA); Bax (Cellular Signaling Technology, MA, USA); p-PI3K (Cellular Signaling Technology, MA, USA); PI3K (Cellular Signaling Technology, MA, USA); p-Akt (T450) (Cellular Signaling Technology, MA, USA); p-Akt (S473) (Cellular Signaling Technology, MA, USA); Akt (Cellular Signaling Technology, MA, USA); mTOR (Cellular Signaling Technology, MA, USA) and GAPDH (Sigma, MO, USA). All chemicals were obtained from Sigma-Aldrich (MO, USA).
4
Immunohistochemical Analysis of ECM Proteins in Efemp1 Mutant Mice
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Eyes from 9-month-old Efemp1+/+, Efemp1ki/ki, and Efemp1−/− mice were fixed in 4% paraformaldehyde. Immunofluorescence staining of 10-μm frozen sections of mouse eyes was performed as previously described33 (link) using a rabbit polyclonal antibody against MMP-2 (Abcam, Cambridge, MA, USA), MMP-9 (Abcam), TIMP-3 (Abcam), or fibulin-3.33 (link) A goat anti-rabbit IgG Alexa Fluor 488 conjugate was used as a secondary antibody (Invitrogen, Carlsbad, CA, USA). Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Sections were examined and photographed using a Nikon E600 microscope (Melville, NY, USA) equipped with a CCD camera.
Quantification of immunofluorescence was performed using Image J software (National Institutes of Health, Bethesda, MD, USA). Sections from three mice (n = 3) per genotype (Efemp1+/+, Efemp1ki/ki, or Efemp1−/−) were used. Three images taken from three randomly selected areas in each stained section were measured. In each image, the outline of the RPE layer and Bruch's membrane was drawn, and the area, mean fluorescence, and background readings were measured. The total fluorescence was calculated as integrated density subtracting (area of selected sections multiplying mean fluorescence of background readings). Fluorescence from Efemp1ki/ki or Efemp1−/− mice was compared with that in Efemp1+/+ mice using a Student's t-test.
Quantification of immunofluorescence was performed using Image J software (National Institutes of Health, Bethesda, MD, USA). Sections from three mice (n = 3) per genotype (Efemp1+/+, Efemp1ki/ki, or Efemp1−/−) were used. Three images taken from three randomly selected areas in each stained section were measured. In each image, the outline of the RPE layer and Bruch's membrane was drawn, and the area, mean fluorescence, and background readings were measured. The total fluorescence was calculated as integrated density subtracting (area of selected sections multiplying mean fluorescence of background readings). Fluorescence from Efemp1ki/ki or Efemp1−/− mice was compared with that in Efemp1+/+ mice using a Student's t-test.
5
Western Blot Analysis of TIMP and MMP
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For the detection of TIMP and MMP protein expression 15 μg of total protein was separated on a 10 % polyacrylamide gel and transferred to Immobilon-P membranes (Millipore, Schwalbach, Germany). After blocking in PBS supplemented with 5 % skim milk (Sigma-Aldrich) and 0.05 % Tween 20 (Sigma-Aldrich) membranes were incubated overnight at 4 °C with one of the following primary antibodies at the indicated dilutions: MMP2 (1:500) (Abcam, Cambridge, United Kingdom), MMP9 (1:500) (Abcam) TIMP1 (1:250) (Abcam), TIMP2 (1:250) (Abcam), TIMP3 (1:250) (Abcam). After washing three times in PBS containing 0.1 % Tween 20 membranes were incubated for 1 h at room temperature with 5000-fold diluted peroxidase conjugated goat anti-mouse IgG (Santa Cruz). Proteins recognized by the antibody were visualized with luminata forte western blotting substrate (Millipore) according to the manufacturer’s instructions. Signal intensities were quantified by densitometry using Bio-1D software version 15.01 (Vilber Lourmat, Eberhardzell, Germany).
6
Immunohistochemical Analysis of Skin Samples
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Unfixed reconstituted human skin samples were embedded in resin (Shandon Cryomatrix; Thermo Fisher Scientific), frozen in liquid nitrogen, and sectioned at 10 μm thickness (CM1950; Leica, Wetzlar, Germany). Replicated cryosections were stained by immunohistochemistry with rabbit antibodies: CLOCK, TIMP3, C/EBP-α (all from Abcam, Cambridge, United Kingdom), as the primary antibody at 4°C overnight. Then, horseradish peroxidase–conjugated donkey anti-rabbit IgG (Abcam) or Alexa Fluor 488-conjugated goat anti-Rabbit IgG (Thermo Fisher Scientific) was applied as the secondary antibody for 1 h at room temperature. Brown immunoreactivity was visualized with 3, 3′-diaminobenzidine as a chromogen, and fluorescent reactivity was evaluated under a microscope (BX53; Olympus, Tokyo, Japan) equipped with a reflected-fluorescence system. Photomicrographs were taken with the microscope’s digital camera (DP72; Olympus).
7
Membrane Protein Extraction and Analysis
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Western blots were performed on total tissue homogenates and nuclear extracts prepared as previously described34 (link).
For preparation of membrane protein fraction, livers were minced into small pieces and homogenized in cold buffer (250 mM sucrose, 1 mM EDTA, 10 mM Tris HCl buffer, pH7.2 plus protease inhibitors). Nuclei and cell debris were removed by centrifugation at 500 g for 10 min at 4 °C, supernatants were subjected to high-speed centrifugation at 100000 g, 4 °C, for 1 h. Pellets were washed with homogenization cold buffer and recentrifuged at 100000 g, 4 °C, for 1 h. The pellet contains membrane fraction.
The lysates were subjected to SDS-PAGE and immunoblot analysis. The following antibodies were used: TIMP3, TNFα, ADAM17, SREBP2 (Abcam), anti–phospho-Ser473 Akt, total Akt, anti–phospho-Thr172 AMPK, total AMPK, ErbB4 (Cell Signaling Technology, Danvers, MA), NRG1 (Genetex, Irvine, CA), Lamin A/C, Actin (Santa Cruz Biotechnologies, Dallas, TX).
For preparation of membrane protein fraction, livers were minced into small pieces and homogenized in cold buffer (250 mM sucrose, 1 mM EDTA, 10 mM Tris HCl buffer, pH7.2 plus protease inhibitors). Nuclei and cell debris were removed by centrifugation at 500 g for 10 min at 4 °C, supernatants were subjected to high-speed centrifugation at 100000 g, 4 °C, for 1 h. Pellets were washed with homogenization cold buffer and recentrifuged at 100000 g, 4 °C, for 1 h. The pellet contains membrane fraction.
The lysates were subjected to SDS-PAGE and immunoblot analysis. The following antibodies were used: TIMP3, TNFα, ADAM17, SREBP2 (Abcam), anti–phospho-Ser473 Akt, total Akt, anti–phospho-Thr172 AMPK, total AMPK, ErbB4 (Cell Signaling Technology, Danvers, MA), NRG1 (Genetex, Irvine, CA), Lamin A/C, Actin (Santa Cruz Biotechnologies, Dallas, TX).
8
Chondrocyte Differentiation Pathway
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9
Protein Expression Analysis of Cell Lysates
10
Inhibition of TACE, TIMP3, and Caveolae
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TACE inhibitor, metalloproteinase inhibitor 3 (Timp3), and caveolae stability inhibitor, lovastatin, were purchased from Abcam (Eugene, OR, USA) and Sigma-Aldrich, respectively.
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