Anti-α-SMA (cs19245), anti-p-AMPKα (cs2535), anti-caspase-1 (cs24232) and anti-NOD-like receptor thermal protein domain associated protein 3 (NLRP3) (cs15101) were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-CollagenI (ab34710), anti-Tissue Inhibitors of Metalloproteinase 1 (TIMP1) (ab61224), anti-caspase-3 (ab179517), anti-beclin-1 (ab210498), anti-Bcl-2 (ab194583) antibodies and the horseradish peroxidase (HRP)-conjugated goat anti-rabbit (ab97051) were purchased from Abcam (Cambridge, MA, USA). Anti-CollagenI (14695-1-AP), anti-SIRT3 (10099-1-AP), anti-F4/80 (28463-1-AP), anti-caspase-6 (10198-1-AP), anti-caspase-9 (10380-1-AP) and anti-GAPDH (60004-1-Ig) antibodies were purchased from Proteintech (Wuhan, HubChina). Anti-Matrix metalloproteinase-13 (MMP-13) (sc515284) and anti-Interleukin (IL)-1R1 (sc393998) antibodies were purchased from Santa Cruz Biotechnology (Stanta Cruze, CA, USA). Anti-IL-1β (mab4012) antibodies were purchased from R&D Systems (Minneapolis, MN, USA). Alexa Fluor 488 Goat anti-Mouse IgG (A11034) and Alexa Fluor Plus 647 Goat anti-Rabbit IgG (A32727) were purchased from Beyotime Biotechnology (Shanghai, China).
Ab61224
Manufactured by Abcam
Sourced in United States
Ab61224 is an antibody product manufactured by Abcam. It is a laboratory tool used for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Ab205719, by Abcam (3 mentions)
Ab92486, by Abcam (3 mentions)
Ripa buffer, by Beyotime (2 mentions)
Ab205718, by Abcam (2 mentions)
Ab38898, by Abcam (2 mentions)
Ab13556, by Abcam (2 mentions)
Ab34710, by Abcam (2 mentions)
Anti gapdh 60004 1 ig, by Proteintech (1 mentions)
Anti collageni ab34710, by Abcam (1 mentions)
Ab179517, by Abcam (1 mentions)
Ab210498, by Abcam (1 mentions)
Bcl 2 ab194583, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Ab32503, by Abcam (1 mentions)
Ab59348, by Abcam (1 mentions)
Ab92536, by Abcam (1 mentions)
Ab73734, by Abcam (1 mentions)
Ab8245, by Abcam (1 mentions)
Ab2302, by Abcam (1 mentions)
Ecl kit, by Merck Group (1 mentions)
13 protocols using ab61224
1
Antibody Resource for Cellular Analysis
2
Western Blot Analysis of Apoptosis and ECM Markers
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All transfected cells were lysed in RIPA buffer (Beyotime, China) and collected by centrifuging at 14000 rpm at 4°C for 10 min. Total protein concentration was determined using the BCA method (Pierce, Rockford, IL, U.S.A.). Equal amounts of the protein were subjected by sodium dodecyl sulfate (SDS)/polyacrylamide gels and then blotted on to polyvinylidene difluoride membranes (Millipore, U.S.A.) by 5% non-fat milk at room temperature for 1 h. Next, the membranes were incubated with primary antibodies. The membranes were washed and then cultured with secondary antibodies (1:2000, #ab205719, #ab205718, Abcam Cambridge, U.K.) for 1 h at room temperature and visualized by using an enhanced chemiluminescence detection reagent (Pierce). All primary antibodies (B-cell lymphoma 2 (Bcl-2, 1:500, 26 kDa, #ab59348), Bax (1:1000, 21 kDa, #ab32503), cleaved Caspase-3 (1:1000, 17 kDa, #ab2302), TIMP metallopeptidase inhibitor-1 (TIMP-1, 1:500, 123 kDa, #ab61224), matrix metallopeptidase (MMP)-2 (MMP-2, 1:1000, 74 kDa, #ab92536), MMP-9 (1:1000, 95 kDa, #ab73734), p53 (1:500, 53 kDa, #ab26) and GAPDH (1:500, 36 kDa, #ab8245)) were purchased from Abcam.
3
Western Blot Analysis of Inflammatory Markers
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Total protein was extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (Boster Biological Technology Co. Ltd.) containing protease inhibitors. The protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Boster Biological Technology Co. Ltd.). Next, the protein was separated by 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred onto the poly(vinylidene fluoride) membranes. The membrane was then blocked with 5% bovine serum albumin (BSA) at room temperature for 2 hours to avoid non‐specific binding, followed by overnight incubation with the following primary rabbit polyclonal antibodies (Abcam Inc) to TLR2 (ab213676, 1:500), TLR4 (ab13556, 1:500), MYD88 (ab2064, 1:1000), TGF‐β (ab92486, 1:500), MMP9 (ab38898, 1:500), TIMP‐1 (ab61224, 1:1000) and GAPDH (ab181602, 1:5000) at 4°C. Afterwards, horseradish peroxidase‐conjugated secondary antibody goat anti‐mouse (ab205719, 1:2000, Abcam Inc) was added to the membrane and incubated for 1 hour at room temperature. The membrane was visualized using an enhanced chemiluminescence (ECL) kit (EMD Millipore). The greyscale quantification of bands in Western blot images was performed using Image J analysis software with GAPDH as an internal reference.16
4
Western Blot Analysis of Protein Expression
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Total proteins were lysed in radioimmunoprecipitation (RIPA) buffer (Beyotime Institute of Biotechnology, Haimen, China). The concentrations of proteins were detected using the Bicinchoninic Acid (BCA) protein assay kit (Thermo Fisher Scientific, Inc.). Equivalent proteins (40 μg) were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes (Merck KGaA, Darmstadt, Germany). Membranes were incubated with primary antibodies, and secondary antibody (cat no LK2003L, Sungene Biotech Co., Ltd, China). The protein was detected using the enhanced chemiluminescence (ECL) substrate kit (Thermo Scientific Pierce) and ImageJ software (NIH, Bethesda, MD, USA). The results were analyzed by densitometry using Quantity One V4.6.2 software (Bio-Rad, USA). The primary antibodies were anti-TLR4 antibody (1: 1000, Abcam, ab13556); anti-COX-2 antibody (1: 1000, Abcam, ab52237); anti-TIMP-1 antibody (1: 1000, Abcam, ab61224); anti-MMP-2 antibody (1: 1000, cat. no. PF023, Millipore, Darmstadt, Germany); anti-MMP-9 antibody (1: 50, Santa Cruz, cat no. sc-21733); anti-E-cadherin antibody (1: 500, Abcam, ab76055); anti-vimentin antibody (1: 800, Abcam, ab92547); anti-p-p65 antibody (1: 1000, Abcam, ab86299); anti-p65 antibody (1: 2000, Abcam, ab16502); anti-β-actin antibody (1: 1000, Abcam, ab8226).
5
HEK293T, KYSE30, and OE19 Cell Hypoxia
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The cell lines HEK293T were got from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cell lines KYSE30 and OE19 were obtained from fenghbio (Hunan, China). Antibodies for ATF5 (ab184923), VEGFA (ab52917), TIMP1 (ab61224), TWIST1 (ab175430), EGF (ab206423), CD31 (ab134168) and POL II (ab264350) were bought from Abcam (Cambridge, MA, USA). Antibodies for PDK1 (13037), PGK1 (68540), CA9 (5649), CBP (7389), P300 (54062), HIF1α (14179), HIF1β (5537) and flag-tag (2368) were procured from Cell Signaling Technology. The human VEGFA ELISA Kit (EK0539) was purchased from BOSTER Biological Technology (Wuhan, China). Identified primers were synthesized by TSINGKE Biological Technology (Beijing, China). For hypoxic conditions, cells were cultured in a sealed hypoxia chamber with a mixture of 1% O2, 5% CO2, and 94% N2 for 2 h.
6
Erythropoietin and Antifibrotic Effects
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The following experimental reagents and drugs were obtained: adenine (Shanghai Ronghe Pharmaceutical Technology Development Co., Ltd., 151029), anti-EPO antibody (Abcam, ab61224), anti-EPOR antibody (Abcam, ab61162), anti-TGF-β1 polyclonal antibody (AbSci, #AB41494), anti-α-SMA antibody (Boster, BM0002), Siwu granules (Jitai'an (Sichuan) Pharmaceutical Co., Ltd., Chinese medicine prescription: Z20020016), recombinant human erythropoietin injection (Shenyang Sansheng Pharmaceutical Co., Ltd., Chinese medicine prescription: S20010001), MDA Kit (Nanjing Jiancheng Bioengineering Institute, A003-1), CAT Kit (Nanjing Jiancheng Bioengineering Institute, A007-1), NO Kit (Nanjing Jiancheng Bioengineering Institute, A013-2), SOD Kit (Nanjing Jiancheng Bioengineering Institute, A001-3), Trizol (Invitrogen, 103106), reverse transcription kit (Thermo Fisher Scientific, #K1662), amplification kit (Roche, 04913914001), and IL-6 (eBioscience, BMS625) and TNF-α (eBioscience, BMS622) ELISA kits.
7
Immunohistochemical Analysis of Fibrosis Markers
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Immunohistochemical staining was performed with an HRP-Polymer anti-Mouse/Rabbit IHC Kit (GTX83398, Irvine, CA, USA) The sections were deparaffinized, washed in phosphate-buffered saline (PBS, 0.01 mol·L−1, pH 7.2) 3 × 5 min, heated at 100 °C in a microwave oven 6 × 2 min, incubated in 3% H2O2 in deionized water for 10 min to block endogenous peroxides activity, and washed 3 × 5 min with PBS. The sections were then incubated overnight at 4 °C with the following primary antibodies: anti-transforming growth factor-β 1 (TGF-β1) antibody (ab92486, Abcam, Cambridge, MA, USA, 1:500); anti-tissue inhibitor of metalloproteinase-1 (TIMP-1) antibody (ab61224, Abcam, 1:200); anti-collagen I antibody (ab34710, Abcam, 1:500). After washing 3 × 5 min with PBS, the appropriate HRP-polymer anti-mouse/rabbit immunoglobulin G was added to the sections and incubated at 37 °C for 20 min. The sections were then washed 3 × 5 min with PBS, and the color was developed with DAB for 3–5 min. The nuclei were lightly counterstained with hematoxylin.
8
Western Blot Analysis of TGF-β1, SPHK1, and TIMP-1
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RIPA solution (Fisher Scientific) was used to extract total protein from in vitro cultured cells, and protein samples were quantified by BCA method. Then, 20 μg protein was subjected to 10% SDS-PAGE gel electrophoresis, then transferred to a PVDF membrane. Blocking was performed by incubating the membrane with 5% skimmed milk. After that, membranes were washed 3 times with PBS, 10 min each time, followed by incubation with primary antibodies, including rabbit anti-TGF-β1 (1: 2000, ab92486, Abcam), anti-SPHK1 (1: 2000, ab46719, Abcam), anti-TIMP-1 (1: 2000, ab61224, Abcam), and anti-GAPDH (1: 1000, ab9845, Abcam) overnight at 4°C. The next day, membranes were washed 3 times with PBS, 10 min each time, followed by incubation with anti-rabbit IgG-HRP secondary antibody (1: 1000, MBS435036, MyBioSource) at room temperature for 1 h. After washing with PBS for 15 min, ECL method (Sigma-Aldrich, USA) was used for signal development. Relative expression levels of TGF-β1, SPHK1, and TIMP-1 were normalized to endogenous control GAPDH using Image J software. This experiment was performed in triplicate.
9
Immunoblotting Antibodies for Signaling Pathways
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The following antibodies were used in the present study: anti-JAK2 antibody (number 3230; Cell Signaling Technology, Boston, Massachusetts, USA); anti-p38 MAPK antibody (ab197348; Abcam, Cambridge, UK); anti-NF-κBp65 antibody (ab16502; Abcam); anti-phospho-stat3 antibody (number 9145; Cell Signaling Technology, Boston, Massachusetts, USA); anti-STAT3 (number 4904, Cell Signaling Technology, Boston, Massachusetts, USA); anti-TIMP1 (ab61224, Abcam, Cambridge, UK); anti-SOCS3 (ab16030, Abcam, Cambridge, UK); and anti-A2M (ab58703, Abcam, Cambridge, UK).
10
Gelatinases and Inhibitors Expression
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The protein expression of gelatinases i.e., MMP-2 & MMP-9 (Sigma-Aldrich, United States; MAB3308, MAB3309) and their respective inhibitors-TIMP-1 & TIMP-2 (Sigma-Aldrich, United States; MAB3310; Abcam, United States; ab61224) in response to cadmium treatment were analyzed by Western blotting. Equal amounts of crude protein was loaded and resolved by reducing SDS-PAGE. Separated proteins were electroblotted to polyvinylidene difluoride (PVDF) membrane (GE Healthcare Limited, Buckinghamshire). Electroblots were washed with TBST (20 mM Tris-buffered saline and 0.1% Tween-20), blocked and probed with primary antibodies for the targets overnight at 4°C. Detection was done using compatible secondary HRP-conjugated antibodies (Abcam, United States; ab205718, ab205719) and incubated in ECL solution for a minute. Immunoreactive bands were visualized using ECL (ThermoFisher-Scientific, United States; 32209) under SynGene Gel documentation system. Protein expressions was evaluated by densitometric analysis using Image studio Lite (Ver 5.2) and normalized against the corresponding expression of GAPDH. Analysis was presented as a fold change compared to the respective controls.
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