Etoposide (VP-16, E1383), and anti-β-actin antibody (A5441) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Human PDGF-BB (100-14B) was purchased from Peprotech. PD98059(S1177), Z-VAD-FMK (S7023), salubrinal (S2923) and SP600125 (S1460) were obtained from Selleck Chemical. The anti-p21 (SC-397) antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The antibodies against ERK (4695P), p-ERK (4370S), cyclin B1 (4138), cdc2 (9116), PARP (9532S), cleaved PARP (5625S), caspase-7 (9491), caspase-9 (9502S), caspase-3 (9662S), cleaved caspase-3 (9661), p-PERK (3179S), eIF2a (5324P), p- eIF2a (3398P), caspase-12 (2202S), CHOP (5554), IRE1α (3294S), p-ASK1 (3764S), JNK (9258P), p-JNK (4668S), p-c-jun (3270S), Bim (2933S), Bcl-2 (2870S), Bax (5023S), and cytochrome c (11940S) and the horse-radish peroxidase-conjugated anti-mouse and anti-rabbit antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). The antibodies against XIAP (MAB822) and hPERK (AF3999) were purchased from R&D Systems (Abingdon, UK). The antibodies against type I collagen (ab138492), α-SMA (ab124964), MMP13 (ab39012), TIMP1 (ab109125) and BIP (ab21685) were obtained from Abcam (Cambridge, UK).
Ab109125
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Ab109125 is a lab equipment product. It is a single-use item designed for use in scientific research and laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Ab76003, by Abcam (2 mentions)
Ab181602, by Abcam (2 mentions)
Ab37150, by Abcam (2 mentions)
Ab38898, by Abcam (2 mentions)
Sp600125 s1460, by Selleck Chemicals (1 mentions)
Ab21685, by Abcam (1 mentions)
Horseradish peroxidase conjugated anti mouse and anti rabbit antibodies, by Cell Signaling Technology (1 mentions)
Anti β actin antibody a5441, by Merck Group (1 mentions)
Ab39012, by Abcam (1 mentions)
Ab124964, by Abcam (1 mentions)
Ab138492, by Abcam (1 mentions)
Xiap mab822, by R&D Systems (1 mentions)
Dako real envision detection system, by Agilent Technologies (1 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Af5001, by Beyotime (1 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
18 protocols using ab109125
1
Molecular Mechanisms of Etoposide-Induced Apoptosis
2
TIMP1 Immunohistochemistry Protocol
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The slides were prepared as described in our recent report [29 (link)]. The primary antibody was used to detect TIMP1 (ab109125, Abcam). The Dako REAL Envision Detection system (Dako, Glostrup. Denmark) was used to detect the protein expression. Hematoxylin (Merk, Darmstadt. Germany) was used to demonstrate nuclear morphology, and DAB (Dako) was used in conjugation with immunoperoxidase detection systems.
3
Western Blot Analysis of Cellular Proteins
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Whole‐cell lysates of cell lines were prepared using radioimmunoprecipitation buffer (P0013J; Beyotime, Shanghai, China) according to the manufacturer's instructions. The protein concentration was determined by a bicinchoninic acid protein assay kit (23225; Thermo Fisher, Rockford, IL, USA). Subsequently, 40 μg of proteins was used for SDS/PAGE and transferred to poly(vinylidene difluoride) membranes. The membranes were blocked for 2 h in 5% nonfat dry milk in TBST and then incubated with the primary antibodies overnight at 4 °C. After incubation with the secondary HRP‐coupled antibodies for 2 h at room temperature, the membranes were washed with TBST, and the immunosignal was developed with enhanced chemiluminescence reagent and exposed in a ChemiDoc XRS + System (1708265; Bio‐Rad, Hercules, CA, USA). β‐Actin was used as the internal control. The concentrations and sources of primary antibodies are as follows: tissue inhibitor of metalloproteinase 1 (TIMP1; ab109125; Abcam, Cambridge, MA, USA) was 1 : 1000, plasminogen activator inhibitor 1 (PAI1; ab222754; Abcam) was 1 : 800, insulin‐like growth factor‐binding protein 1 (IGFBP1; ab181141; Abcam) was 1 : 1000, IL35 (LAC008Hu72; Cloud‐Clone Corp.) was 1 : 1000 and β‐actin (AF5001; Beyotime) was 1 : 2000.
4
Western Blot Analysis of MMP and TIMP
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Protein was extracted from fresh NIP tissue using ice-cold RIPA lysis buffer (Abcam) mixture containing protease and phosphatase inhibitor (Sigma Aldrich). After 30 minutes of incubation on ice with shaking, followed by centrifugation at 14,000 rpm, the supernatant containing the protein was aspirated for use in Western blot analysis. Protein concentration was first determined using BCA protein assay (Thermo Fisher, USA); then, 4X laemmli sample buffer was added to the protein samples according to the manufacturer’s instructions, and heated at 95°C for 10 minutes. 30 μg total protein from each sample was subjected to electrophoresis in 10% SDS-PAGE gel and then transferred onto a 0.22μm PVDF membrane (Millipore, USA). After 2 hours non-specific blocking with 5% skimmed milk, primary antibody of MMP-1 (1:2000, Abcam, ab137332), MMP-7 (1:2000, Abcam, ab207229), MMP-9 (1:4000, Abcam, ab76003), TIMP-1 (1:2000, Abcam. Ab109125), TIMP-3 (1:2000, Abcam, ab39184), GAPDH (1:5000, Abcam, ab186930) were added to the membrane and incubated at 4°C overnight. HRP-conjugated anti-rabbit IgG or anti-mouse IgG were then added as secondary antibody at room temperature for 1 hour. After secondary antibody staining, ECL reagent (Bio-Rad, USA) was used to visualize the blots. Densitometry was performed with ImageJ software to quantify the protein amounts of each sample.
5
Protein Expression Detection by Western Blot
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The total protein of the cell lysates was extracted after treatment with different conditions. A detailed description of the preparation is presented in our previous report [29 (link)]. The primary antibodies used to detect specific proteins were β-actin (A544, Sigma), LC3 (PM036, Medical and Biological Laboratories, Nagoya, Japan), TIMP1 (ab109125, Abcam, Cambridge, UK), Rab37 (LTK BioLaboratories, Taiwan), ATG5 (ab108327, Abcam), and ATG7 (ab133528, Abcam). Samples were incubated overnight at 4 °C. The membranes were incubated with the secondary anti-rabbit (Amersham Pharmacia, Piscataway, NJ, USA) or anti-mouse (Chemicon, Temecula, CA, USA) antibody at room temperature for 1 h. Finally, the membrane was rinsed with enhanced chemiluminescence (ECL) (WBKLS0500; Millipore) and exposed using the BioSpectrum AC system (101-206-009; UVP, Upland, CA, USA).
6
Protein Expression Analysis via Western Blot
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Western blot (WB) assays was performed as previously described (30 (link)). Briefly, we prepared cell extracts for Western blotting in RIPA buffer. Then, lysates were separated by SDS-PAGE and were transferred to PVDF membranes (Millipore, Billerica, MA). Primary antibodies PDPN (Abcam, ab236529,1:1000), TIMP1 (Abcam, ab109125,1:1000), EMP3 (Santa cruz, sc-81797, 1:100), TAGLN2 (Proteintech, 10234-2-AP, 1:200), and GAPDH (Abcam, ab181602, 1:10000) were used along with HRP-labeled secondary antibody (1:10000, Sigma) in Western blot. The immune complex was detected by chemiluminescence (GE Healthcare, Wauwatosa, WI).
7
Protein Extraction and Western Blot Analysis
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Total proteins were extracted using a Total Protein Extraction Kit (KeyGEN, Nanjing, China) and quantified using a bicinchoninic acid (BCA）protein assay kit (Beyotime, Shanghai, China). Equal amounts of proteins (20‐30 µg) were separated by 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis, and then electrotransferred onto 0.45 or 0.2 µm polyvinylidene difluoride membranes (Millipore). After blocking with 5% skim milk, the membranes were incubated overnight at 4°C with primary antibodies (1:1000) and at room temperature for 2 hours with secondary antibodies (1:5000). Protein bands were detected by chemiluminescence (Thermo, MA, USA). The following primary antibodies were used: KDM1A (CST, #2184), TIMP1 (abcam, Shanghai, China, ab109125), MMP9 (abcam, ab76003), MMP2 (abcam, ab37150), H3K4me2 (abcam, ab32356), H3K9me2 (abcam, ab176882), GAPDH (Sigma‐Aldrich, MO, USA #G9545) and total histone H3 (CST, #4499).
8
Immunofluorescence analysis of TIMP1, LC3, and Rab37
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The cells were seeded onto a 2-well chamber slide (SPL, Korea) and treated with different conditions. Anti-TIMP1 (ab109125, Abcam), anti-LC3 (PM036, M186-3, MBL), or anti-Rab37 (LTK BioLaboratories, Taiwan) antibody was used. The fluorescent change of the cells was detected under a multi-photon confocal microscope (Olympus, FV1000MPE, Tokyo, Japan). A detailed description is provided in our previous report [29 (link)].
9
Western Blot Analysis of TIMP1, Fn1, GAPDH
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The treated cells were lysed. After determination of the protein concentration, a total of 25 µg protein was separated and transferred to PVDF membranes (Millipore, Bedford, USA). After blocking, the membrane was incubated overnight with the primary antibodies at 4 °C. The proteins were detected using an enhanced chemiluminescence (ECL) system. The antibodies used are as follows: Rabbit antibodies against TIMP1, Fn1, and GAPDH (ab109125, ab2413, and ab181602, 1:2000, Abcam, Cambridge, MA, USA).
10
Protein Expression Analysis of RCC Tissues
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Total protein was extracted from RCC tissues and corresponding adjacent normal tissues of 12 patients using RIPA lysis buffer (Servicbio.Inc.) with protease inhibitor phenyl methane sulfonyl fluoride (PMSF, 1%), and then the concentration was measured with BCA protein assay kit (Beyotime Biotechnology, Jiangsu, China). Primary rabbit polyclonal antibody against primary antibodies (TIMP1 1:1,000, Abcam,Inc., ab109125; N-cadherin 1:5000, Abcam.Inc., ab76011; E-cadherin 1:10000, Abcam,Inc., ab40772) and β-actin (1:10000; Abclonal,Inc., cat.AC026) were incubated overnight at 4°C. All the procedures were performed according to the manufacturer’s instructions.
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