The recombinant protein for human TGF-β1 (#P01137) and mouse monoclonal antibodies against human TIMP-1 (#MAB970), TIMP-3 (#MAB973), MMP-2 (#MAB9022), and MMP-9 (#MAB911) were acquired from R&D Systems, Inc (MN, USA). The rabbit polyclonal antibodies against CTGF (#ab6992), fibronectin (#ab2413), and α-SMA (#ab5694) were acquired from Abcam Inc. (Cambridge, UK). The mouse monoclonal antibodies against p38 (#ab31828), p38 phosphorylation (p-p38, #ab4822), JNK (#ab208035), JNK phosphorylation (p-p38, #ab76572), ERK (#ab184699), and ERK phosphorylation (p-p38, #ab201015) were all acquired from Abcam Inc. (Cambridge, UK). The secondary antibodies against rabbit (#A5795) and mouse (#A9044), as well as GAPDH (#G5262) and β-actin (#A5441), were acquired from Sigma-Aldrich (St. Louis, MO, USA) [6 (link)].
Ab76572
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab76572 is a lab equipment product manufactured by Abcam. It serves as a tool for scientific research and experimentation. The core function of this product is to perform a specific task within a laboratory setting. No further details about its intended use or applications can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Ab208035, by Abcam (7 mentions)
Ab184699, by Abcam (6 mentions)
Ab4822, by Abcam (4 mentions)
Ab201015, by Abcam (4 mentions)
Ab179461, by Abcam (4 mentions)
Ab31828, by Abcam (3 mentions)
Ab170099, by Abcam (3 mentions)
Ab6992, by Abcam (2 mentions)
A9044, by Merck Group (2 mentions)
Ab2413, by Abcam (2 mentions)
Mab970, by R&D Systems (2 mentions)
A5795, by Merck Group (2 mentions)
Ab5694, by Abcam (2 mentions)
Ab6721, by Abcam (2 mentions)
P erk, by Cell Signaling Technology (2 mentions)
Ab178867, by Abcam (2 mentions)
Ab205718, by Abcam (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
G5262, by Merck Group (1 mentions)
14 protocols using ab76572
1
Antibody Panel for TGF-β1 and MMPs
2
Salivary Gland Protein Expression Analysis
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Total proteins were extracted from salivary glands tissues using RIPA lysis buffer. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (10%) was used for protein electrophoresis. The proteins were then transferred to the polyvinylidene difluoride (PVDF) membranes. After blocking with 5% bovine serum albumin, the membranes were incubated with diluted primary antibodies for 12 h at 4°C. After washing, the PDVF membranes were incubated with diluted goat anti-rabbit IgG H&L (HRP) (1:5,000, ab6721; Abcam), goat anti-mouse IgG H&L (HRP) (1:5,000, ab205719; Abcam) at room temperature for 2 h. The bands were detected using the ECL system (Bio-Rad) and analyzed using Image J. The primary antibodies used were p-NF-κB (1:1,000, ab239882; Abcam), NF-κB (1:1,000, ab207297), p-ERK (1:1,000, ab201015), ERK (1:10,000, ab184699), p-JNK (1:5,000, ab76572), JNK (1:1,000, ab179461), p-P38 (1:1,000, ab45381), P38 (1:1,000, ab170099), and β-actin (1 µg/ml; ab8226).
3
YfeA modulates signaling pathways in RAW 264.7 macrophages
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RAW 264.7 macrophages were treated with or without YfeA (20 µg/mL) for 12 h, the total cellular protein was extracted using a Protein Extracting Kit (Solarbio, Beijing, China). Aliquots corresponding to 50 µg of each sample were analyzed using Western blotting (WB). To analyze the YfeA-induced signal transduction residue phosphorylation, the primary monoclonal antibodies, including Erk1/2 (rabbit, 42/44 kDa; 1:2000; Abcam ab184699), p-Erk1/2 (rabbit, p-ERK1: Thr202/Tyr204, p-ERK2: Thr185/Tyr187; 42/44 kDa; 1:2000; Abcam ab278538), p38-MAPK (rabbit, 41 kDa; 1:1000; Abcam ab31828), p-p38-MAPK(rabbit, Tyr182; 41 kDa; 1:500; Abcam ab47363), JNK (rabbit, 48 kDa; 1:1000; Abcam ab179461), p-JNK (rabbit, Tyr185/223; 48 kDa; 1:10,000 Abcam ab76572), p65 (NF-κB; rabbit, 60 kDa; 1:2000; Abcam ab16502), p-p65 (NF-κB; rabbit, Ser536; 60 kDa; 1:5000; Abcam ab86299), TLR2 (rabbit, 89 kDa; 1:500; Abcam ab213676), and TLR4 (rabbit, 89 kDa; 1:500; Abcam ab13556) were applied at this stage.
4
Western Blot Analysis of Signaling Pathways
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RL95-2 cells were treated and the lysate was prepared as described previously (23 (link)). The protein (20 µg) was separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (EMD Millipore; Billerica, MA, USA). Immunoblots were exposed to primary antibodies at room temperature for 1 h. Antibodies used were against p38 (9212; 1:2,000 dilution), phosphorylated (p)-p38 (4511l 1:1,000 dilution), NF-κB p65 (8242l 1:1,500 dilution), p-p65 (3033l 1:2,000 dilution), extracellular signal-regulated kinase (ERK; 4695; 1:1,000 dilution), p-ERK (4370l 1:1,500 dilution) (all from Cell Signaling Technology, Inc.), c-Jun N-terminal kinase (JNK; ab179461; 1:1,000 dilution) or p-JNK (ab76572; 1:1,500 dilution) (all from Abcam, Cambridge, MA, USA). Subsequently, blots were incubated at room temperature for 40 min with horseradish peroxiase-conjugated secondary antibodies (BA1054; 1:20,000 dilution; Boster Biological Technology, Wuhan, China). The bands with immunoreactive proteins on the membrane were assessed by the ECL Plus Western Blotting Detection system (EMD Millipore). GAPDH was selected as the reference protein (kC-5G5; 1:10,000 dilution; Kangchen Biotech Co., Ltd., Shanghai, China).
5
Molecular Regulation of Osteoclastogenesis
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MedChemExpress (New Jersey, USA) provided the high purity (≥99.0%) SAHA used in this study. Dimethyl sulfoxide (DMSO) (Sigma–Aldrich, Sydney, NSW, Australia) was employed to prepare SAHA to a concentration of 10 mM for storage at − 20 °C before being diluted to working concentrations with culture medium. Dulbecco’s modified Eagle’s medium (DMEM/high glucose) along with fetal bovine serum (FBS) were acquired from HyClone (Logan, UT, USA), whereas recombinant murine RANKL was supplied by R&D system (Minneapolis, MN, USA). FITC-phalloidin was procured from Thermo Fisher Scientific (Scoresby, VIC, Australia) and DAPI staining solution was bought from Beyotime (Shanghai, China). Primary antibodies against β-Actin (AC006), NFATc1 (A1539), CTSK (A5871), MMP9 (A11147), phospho-P38 (AP0057), phospho-ERK (AP0485), ERK (A4782), and phosphor-P65 (AP0475) were obtained from ABclonal (Wuhan, China). Primary antibody against IκB-α (AF5002) was obtained from Affinity Biosciences (Jiangsu, China). Primary antibodies for P38 (ab170099), phospho-JNK (ab76572), JNK (ab208035), and TRAF6 (ab40675) were purchased from Abcam (Cambridge, UK). The corresponding secondary antibodies were purchased from Beyotime (Shanghai, China). Yangming Biotechnology (Hangzhou, China) supplied bovine bone slices.
6
Protein Extraction and Western Blot Analysis
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Tissues or cells were lysed in RIPA lysis buffer (Beyotime) to extract total proteins. After separation by 12% SDS‐PAGE, proteins were transferred onto PVDF membranes (Bio‐Rad) and then blocked in 5% skim milk. The membranes were next incubated with the primary and secondary antibodies, including anti‐MAP4K3 (#92427; Cell Signaling Technology), antiproliferating cell nuclear antigen (anti‐PCNA; ab18197; Abcam), anti‐Bcl‐2‐associated X protein (anti‐Bax; ab182733), anti‐ERK1/2 (ab17942; Abcam), anti‐p‐ERK1/2 (ab223500; Abcam), anti‐JNK (AB208035; Abcam), anti‐p‐JNK (ab76572; Abcam), anti‐p38 (ab32142; Abcam), anti‐p‐p38 (ab178867; Abcam), anti‐GAPDH (ab9485; Abcam) and goat‐anti rabbit (ab205718; Abcam). The protein signals were detected using an enhanced chemiluminescence system (Beyotime).
7
Evaluation of Antioxidant and Anti-inflammatory Effects
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The MO was obtained from Hainan Academy of Agricultural Sciences (Hainan, China). The absolute alcohol was purchased from Xilong Science Co., Ltd. (Shantou, China). The enzyme-linked immunoassay assay (ELISA) kits for reactive oxygen species (ROS) and myeloperoxidase (MPO) were purchased from Shanghai X-Y Biotechnology Co., Ltd. (Shanghai, China). The assay kits for malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) were purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). The primers (Nrf2, Keap1, HO-1, p38, JNK and ERK) were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). The antibodies (Nrf2: ab92946, Keap1: #8047S, HO-1: ab189491, p38: ab4822, p-p38: ab4822, JNK: ab179461, p-JNK: ab76572, ERK: ab184699 and p-ERK: ab201015) were purchased from Abcam (Cambridge, UK) and Cell Signaling Technology (Boston, MA, USA).
8
Immunoblotting Protein Expression Analysis
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Cells were lysed in RIPA lysis buffer (Applygen, Beijing, China) containing protease inhibitors (Applygen, Beijing, China) and phosphatase inhibitors (Applygen, Beijing, China). Protein concentrations were measured using a BCA kit (Applygen, Beijing, China). After sodium dodecyl sulfate-polyacrylamide gel electrophoresis for 45 min at a constant voltage of 80 V and 45 min at 120 V, the separated proteins were transferred to PVDF membranes (Millipore, Sigma, Billerica, MA, USA). The membranes were blocked with 5% skimmed milk powder at room temperature for 1.5 h and then incubated overnight at 4 °C with primary antibodies: rabbit anti-ADAM9 mAb (1:1000, 4151, Cell Signaling Technology, Danvers, MA, USA), mouse-anti-IL-17A mAb (1:1000, 66148-1-lg, Proteintech, Rosemont, IL, USA), rabbit anti-JNK mAb (ab208035, Abcam, Cambridge, MA, USA), and rabbit anti-phospho-JNK mAb (ab76572, Abcam, Cambridge, MA, USA). HRP-labeled goat anti-rabbit or goat anti-mouse lgG (ZSGB-Bio, Beijing, China) was used as secondary antibodies. Immunoreactive bands were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore, Sigma, Billerica, MA, USA) and analyzed using ImageJ software (National Institutes of Health, MD, USA).
9
Profibrotic Signaling Pathway Modulators
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Pirfenidone (#P2116) and the secondary antibodies against rabbit (#A5795) and mouse (#A9044) were acquired from Sigma-Aldrich (St. Louis, MO, USA). The human TGF-β1 recombinant protein (#P01137) and the mouse monoclonal antibodies against TIMP-1 (#MAB970) were acquired from R&D Systems, Inc. (Minneapolis, MN, USA). Antibodies including the rabbit polyclonal antibodies against CTGF (#ab6992), fibronectin (#ab2413), collagen type I (#ab34710), and α-SMA (#ab5694), the mouse monoclonal antibodies against p38 (#ab31828), p38 phosphorylation (p-p38, #ab4822), JNK (#ab208035), and JNK phosphorylation (p-p38, #ab76572) were all purchased from Abcam Inc. (Cambridge, UK).
10
Dapagliflozin and Ferroptosis Inhibition
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Dapagliflozin was obtained from MCE (Cat. HY-10450). Ferrostatin-1 (Fer-1) was purchased from Selleck Chemicals (Cat# S7243). Drugs were separately dissolved in DMSO and added to the culture medium 12 h before H/R. For in vivo experiments, DAPA was purchased from commercially available dapagliflozin tablets (Forxiga Tab 10 mg, AstraZeneca AB, United States). DAPA was diluted with sterile water for injection (WFI) and given to animals by intragastric administration, as previously shown (Nikolaou et al., 2022 (link)). Primary antibodies against β-tubulin (1:5,000, Cat. AC021), GPX4 (1:1,000, A1933), SLC7A11 (1:1,000,A2413), ACSL4 (1:1,000, A6826), PTGS2 (1:1,000, A1253), and FTH1 (1:1,000, A19544) were purchased from ABclonal (Wuhan, China); FTMT (1:200,aa65-227) was purchased from LifeSpan BioSciences (Seattle, WA, United States); p-ERK (1:1,000,#4370), ERK (1:1,000, #4695), p-P38 (1:1,000, #4511), and P38 (1:1,000, #8690) were purchased from Cell Signaling Technology (Beverly, MA, United States); p-JNK (1:1,000, ab76572)), and JNK (1:1,000, ab208035) were purchased from Abcam (San Francisco, CA).
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