OC precursors were stimulated by RANKL with or without vimentin, CV, Rottlerin (Protein kinase C (PKC)-δ inhibitor; Thermo Fisher Scientific, Waltham, MA, USA), KT 5720 (PKA inhibitor, Santa Cruz Biotechnology, Dallas, TX, USA), anti-vimentin mAb (mouse IgM, clone 3CB2) or isotype control mAb (mouse IgM, clone TEPC183, Ancell Corp., Stillwater, MN, USA) for 7 days with the addition of a fresh medium change every 3 days. TRAP staining was performed with a TRAP staining kit (Sigma-Aldrich) according to the manufacturer’s protocol. Briefly, the cells were fixed by a citrate (0.38 mol/L)/acetone solution for 30 s at room temperature. After washing with deionized water, the cells were stained with a TRAP staining solution (L(+)-tartrate buffer, 0.67 mol/L; acetate buffer, 2.5 mol/L; Naphthol AS-BI phosphoric acid, 12.5 mg/mL; and Fast Garnet GBC base, 7.0 mg/mL ) for 10 min at 37 °C in the dark. Cells with ≥ 3 nuclei were determined as multinucleated OCs.
Kt5720
Manufactured by Santa Cruz Biotechnology
Sourced in Germany
KT5720 is a small molecule inhibitor that selectively inhibits the activity of cAMP-dependent protein kinase (PKA). It acts by binding to the catalytic subunit of PKA, thereby inhibiting its enzymatic activity.
Automatically generated - may contain errors
Lab products found in correlation
Trap staining kit, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium nutrient mixture f 12 dmem f 12, by Thermo Fisher Scientific (1 mentions)
Bca kit, by Beyotime (1 mentions)
Gw5074, by Selleck Chemicals (1 mentions)
Pe conjugated monoclonal active caspase 3 antibody apoptosis kit, by BD (1 mentions)
Anti rabbit secondary antibody conjugated to horseradish peroxidase, by Santa Cruz Biotechnology (1 mentions)
Primary antibodies against erk1 2 and phospho erk1 2, by Cell Signaling Technology (1 mentions)
Nesfatin 1, by Phoenix Pharmaceuticals (1 mentions)
Pd98059, by Beyotime (1 mentions)
Anti brdu antibody, by BD (1 mentions)
Brdu staining kit, by BD (1 mentions)
Capsaicin, by Merck Group (1 mentions)
Nifedipine, by Merck Group (1 mentions)
Morphine sulphate pentahydrate, by Merck Group (1 mentions)
Forskolin, by Biotrend (1 mentions)
3 isobutyl 1 methyl xanthine (ibmx), by Merck Group (1 mentions)
Verapamil hydrochloride, by Merck Group (1 mentions)
Menthol, by Merck Group (1 mentions)
Naloxone hydrochloride dihydrate, by Merck Group (1 mentions)
Phorbol 12 myristate 13 acetate pma, by Merck Group (1 mentions)
8 protocols using kt5720
1
Osteoclastogenesis Regulation by Vimentin
2
Nesfatin-1 Induced Apoptosis Pathway
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Unless otherwise stated, all chemicals were purchased from Sigma Chemical Co. (St. Louis. MO, USA). MES23.5 cells were provided by Professor Wei-Dong Le (Baylor College of Medicine, TX, USA). Nesfatin-1 was obtained from Phoenix Pharmaceuticals Inc. (Burlingame, CA, USA). Dulbecco’s modified Eagle’s medium nutrient mixture-F12 (DMEM/F12) was purchased from Gibco (Grand Island, NY, USA). The PE-conjugated monoclonal active-caspase-3 antibody apoptosis kit was from BD Bioscience (San Diego, CA, USA). PD98059 and the BCA kit was from Beyotime (Shanghai, CN). KT5720 and anti-rabbit secondary antibody conjugated to horseradish peroxidase were from Santa Cruz (Santa Cruz, CA). The cell fractionation kit was from Clontech (Mountain View, CA, USA). Anti-Cox IV monoclonal antibody was from Clontech (CA, USA). The primary antibody against TH was from Millipore (Darmstadt, Germany). Primary antibodies against ERK1/2 and phospho-ERK1/2 were from cell signaling technology (MA, USA). Alexa Fluor 555 donkey anti-rabbit secondary antibody was from Eugene (OR, USA). GW5074 was from Selleck (TX, USA).
3
Antibody Sourcing and Reagent Procurement
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Polyclonal and monoclonal antibodies (PcAb and McAb) were purchased from commercial sources: PcAb against phospho(p)-ERK1/2, ERK1/2, and McAb against HA were from Cell Signaling Inc.; McAb against murine COL2α1 was from Thermo Scientific; PcAb against MMP13, SOX9 and COL10α1 were from Abcam;. PcAb against SUMO1, IHH, and SHP2 were purchased from Invitrogen, Epitomics and UBI, respectively. PcAb against β-CATENIN were purchased from Cell Signaling (MA). Anti-BrdU antibodies and BrdU staining kit were obtained from BD pharmingen. PE-conjugated, affinity purified PcAb against SOX9 were purchased from Bioss. The PKA inhibitor KT5720 was purchased from Santa Cruz. PE-conjugated anti-Rabbit IgG was purchased from Cell Signaling. Alcian blue and Alizarin Red S staining solutions were purchased from Poly Scientific.
4
Preparation and Use of Chemical Reagents
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All chemical reagents were prepared as stock solutions in DMSO, with the exception of PTX (pertussis toxin from Bordetella pertussis) and morphine that were dissolved in H2O. Stocks were kept aliquoted and frozen at -20°C. Even when several compounds were applied simultaneously, the final DMSO concentration in the superfusing solution did not exceed 0.4%. The following substances were obtained from Sigma-Aldrich: AITC (allyl-isothiocyanate), capsaicin, DAMGO ([D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin acetate), menthol, morphine sulphate pentahydrate, IBMX (3-Isobutyl-1-methylxanthine), naloxone hydrochloride dihydrate, nifedipine, verapamil hydrochloride. The following substances were purchased from Biotrend: forskolin, herkinorin, loperamide hydrochloride, (RS)-baclofen, WIN 55,212-2, somatostatin-14, DHPG (RS-3,5 dihydroxyphenylglycine), [D-Ala2] deltorphin II. L-noradrenaline was obtained from Alfa Aesar (Thermo Fisher), BIM (Bisindolylmaleimide IV) was from Biomol (Hamburg, Germany), H89 dihydrochloride hydrate from Biozol (Eching, Germany), KT5720 from Santa Cruz (Heidelberg, Germany) and pregnenolone sulfate (PS) from Steraloids (Newport, RI, USA). The cell-permeable peptides mSIRK and mSIRK-9LA were purchased from Calbiochem (Merck-Millipore, Darmstadt, Germany).
5
Inhibition of JH-III in Cell Culture
6
Ringer's Solution Preparation Protocol
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Alamandine was purchased from Phoenix Pharmaceuticals, Inc.; D-Pro7-Ang(1-7) and A-779 were obtained from Bachem; L-NMMA was obtained from Sigma–Aldrich; and KT5720 was obtained from Santa Cruz Biotechnology. All drugs were prepared in double-distilled water, with the exception of KT5720 prepared in DMSO. All dilutions were made in the Ringer’s solution immediately before use.
7
Reagents and Chemicals for Cell Experiments
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U73343 and U73122 were from Tocris Bioscience (Bristol, UK). Staurosporine was from Enzo Life Sciences (Exeter, UK). KT 5720 was from Santa Cruz Biotechnology (Heidelberg, Germany). Jasplakinolide and LIM kinase inhibitor 1 LIMKi3 were from Calbiochem (Nottinghamshire, UK). FM 1-43 was from Invitrogen (Paisley, UK). Glutaraldehyde, 25% EM Grade was from Agar Scientific (Essex, UK). All other reagents, including HPTS were from Sigma-Aldrich (Dorset, UK).
8
Synthesis of CDNB-Glutathione Conjugate
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Bovine serum albumin (BSA), 1-chloro-2,4-dinitrobenzene (CDNB), dibutyryl cyclic-AMP sodium salt (db-cAMP), estradiol-17β-d-glucuronide (E 2 17G), Gö6976, leupeptin, MK571, pepstatin A and phenylmethylsulfonyl fluoride (PMSF) were from Sigma-Aldrich. KT5720 was from Santa Cruz Biotechnology. DMSO was from Merck. All other chemicals were of analytical grade purity. The glutathione-conjugated derivative of CDNB, 2,4-dinitrophenyl-S-glutathione (DNP-SG), was synthesized with the use of 1-fluoro-2,4-dinitrobenzene and glutathione as described by Sokolovsky et al. (1964) .
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