293T and WI-26 cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin in 5% CO2 at 37 °C. WI-38, A549, H460, HCC827, and H1650 cells were cultivated in RPMI as described above. All of the cells used and GFP-tagged ERK were gifted from the Biocon biobank (Seoul National University, Seoul, Korea). GFP-HSP70 (#15215) was purchased from Addgene and subcloned into the EcoRI/XhoI sites of pEXPR-IBA105. Each of the HSP70 domains was cloned into the EcoRI/XhoI sites of pEGFP-C3, pEGFP-N1 and pGEX4T-1. The point mutagenesis of HSP70 was performed with a QuikChangeII kit (Promega) according to the manufacturer’s instructions. EGF was purchased from Peprotech. MG-132 and antibody against p-Ser were purchased from Sigma. Antibodies against p-Thr, p-Tyr, p-ERK, and ERK were purchased from Cell Signaling Technology, and other antibodies were purchased from Santa Cruz Biotechnology. StrepMAB-classic-HRP for detecting Strep was purchased from IBA Lifesciences. U0126 (#1144, Tocris), SP600125 (#1496, Tocris), SB203580 (#8307, Sigma) and Fludarabine (#F9813, Sigma) were purchased from the respective suppliers. LY294002 (#440202) was purchased from MERK. siRNA against ERK was purchased from Invitrogen (#VHS40404 and #VHS40318).
Other antibodies
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Other antibodies are proteins produced by the immune system that can recognize and bind to specific target molecules. These antibodies are used in various laboratory applications, such as immunoassays and protein detection. They serve as tools for researchers to study biological processes and identify specific proteins or other analytes of interest.
Automatically generated - may contain errors
Lab products found in correlation
Fludarabine, by Merck Group (1 mentions)
Sp600125, by Bio-Techne (1 mentions)
Sb203580, by Merck Group (1 mentions)
Gfp hsp70, by Addgene (1 mentions)
Ly294002, by Merck Group (1 mentions)
U0126, by Bio-Techne (1 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Spermine, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by GE Healthcare (1 mentions)
Calhex231, by Merck Group (1 mentions)
Fura 2 acetoxymethyl ester am, by Thermo Fisher Scientific (1 mentions)
Skf96365, by Merck Group (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Tissue culture dishes, by BD (1 mentions)
Culture media, by Thermo Fisher Scientific (1 mentions)
Standard reagent, by Merck Group (1 mentions)
Fura 2 am, by Thermo Fisher Scientific (1 mentions)
Prostaglandin e2, by Cayman Chemical (1 mentions)
Anti fcp1 antibodies, by Fortis Life Sciences (1 mentions)
8 protocols using other antibodies
1
Cell Culture and Protein Analysis Methods
2
Regulation of Calcium Signaling in Cells
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Fetal bovine serum (FBS) was obtained from HyClone; GE Healthcare Life Sciences (Logan, UT, USA), and all other cell culture reagents were purchased from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Spermine (a CaSR agonist), Calhex231 (a CaSR negative allosteric modulator), MRS1845 (a SOCC inhibitor) and SKF96365 (a TRPC inhibitor) were obtained from Sigma-Aldrich; Merck Millipore (Darmstadt, Germany). Rabbit anti-TRPC1 monoclonal antibody (catalog no. ACC-010) was obtained from Alomone Laboratories, Ltd. (Jerusalem, Israel). Polyclonal mouse anti-human CaSR antibody was obtained from Abcam (Cambridge, MA, USA; catalog no. ab62653, for western blotting) and from Shanghai Seebio Science & Technology Co., Ltd. (Shanghai, China; catalog no. HL1499 for immunohistochemistry) and other antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Small interfering RNA (siRNA) was purchased from Yangzhou Ruibo Biotech Co., Ltd. (Yangzhou, China). Lipofectamine™ 2000, Fura-2-acetoxymethyl ester (AM) and the NO Fluorescence kit were obtained from Invitrogen; Thermo Fisher Scientific, Inc.
3
Signaling Mechanisms of Purinergic Receptor Activation
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UTP, ionophores, and standard reagents were from Sigma-Aldrich (St Louis, MO, USA). DFU was from Merck (Rahway, NJ, USA). Prostaglandin E2 was from Cayman Chemical (Ann Arbor, MI, USA). Gö6976, Gö6983, Gö6850, and inhibitors of standard signaling pathways were from Calbiochem (San Diego, CA, USA). Fura-2/AM was from Invitrogen (Carlsbad, CA, USA). Cytokines were from PeproTech (London, UK). Antibodies against P2Y2, P2Y4, and P2Y6 receptors were from Alomone Labs (Jerusalem, Israel) and other antibodies were from Santa Cruz Biotech (Santa Cruz, CA, USA), from Cell Signaling (Danvers, MA, USA), or from the sources previously described [22 ]. Reagents for electrophoresis were from Bio-Rad (Hercules, CA, USA) and Sigma-Aldrich. Tissue culture dishes were from Falcon (Lincoln Park, NJ, USA) and culture media were from Invitrogen.
4
Protein Immunoblot and Immunoprecipitation Protocol
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Anti-P-Y15-cdk1, MPM-2 and Flag antibodies were from Cell Signaling Technologies (Danvers, MA, USA), Merck Millipore (Billerica, MA, USA) and Sigma-Aldrich respectively. Anti-Fcp1 antibodies were from Bethyl Laboratories (Montgomery, TX, USA) and Santa Cruz Biotechnology (Dallas, TX, USA). Rabbit polyclonal antibodies against P-T239 of human Wee1 were previously described.10 (link) Other antibodies were from Santa Cruz Biotechnology. Immunoprecipitations and immunoblots were performed as described.10 (link) Densitometric quantitations of immunoblot signal intensity were performed by using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).
5
Dissection of ONC201 Signaling Pathway
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ONC201 (TIC10) was obtained from Selleck (Shanghai, China); The pan caspase inhibitor z-VAD-fmk and the caspase-8 inhibitor z-IETD-fmk were from CalBiochem (La Jolla, CA). The kinase antibodies utilized in this study were purchased from Cell Signaling Tech (Shanghai, China). Other antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Cell culture reagents were provided by Gibco (Shanghai, China).
6
Western Blot Analysis of Epithelial-Mesenchymal Markers
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HMCs were lysed in radioimmunoprecipitation assay buffer containing a protease inhibitor cocktail (Roche). Total protein of 40 μg was isolated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride (PVDF) membranes (Roche). After blocking with tris buffered saline tween buffer with 5% skim milk, the PVDF membranes were incubated with primary antibodies. Thereafter, the PVDF membranes were incubated with second antibodies. The primary antibodies were exhibited as follows: anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab9484), anti-LIN28B (ab115698), anti-N-cadherin (sc-8424), anti-E-cadherin (sc-8426), anti-collagen IV (sc-59814), and anti-fibronectin (sc-18825). Protein bands were visualized with an ImmunoStar LD (Wako Pure Chemical). The primary antibodies for LIN28B and GAPDH were bought from Abcam, and other antibodies were purchased from Santa Cruz Biotechnology. All original protein bands of western blotting were exhibited in Supplementary Fig. 2 (available online).
7
Preparation and use of melanogenic compounds
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Dulbecco s Modified Eagle s Medium (DMEM) and RPMI1640 were obtained from Nissui Pharmaceutical (Tokyo, Japan) . Fetal bovine serum was obtained from Gibco (Gaithersburg, MD, USA) . Antibody against MITF was purchased from Cell Signaling Technology (Beverly, MA, USA) . Other antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) . α-MSH was purchased from Sigma-Aldrich (St. Louis, MO, USA) . Methyl eugenol, acetyl isoeugenol, isoeugenol, and 2-methoxy-4-ethylphenol were obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan) . Chavicol was were purchased from Nacalai tesque Inc. (Kyoto, Japan) . All other chemicals were obtained from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) . Before use, eugenol and structurally related compounds were dissolved in ethanol and stored at -20℃.
8
Cell Line Preparation and Reagents
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Chemical preparations and cell lines. UA, with a purity of 98.6%, was obtained from Xi'an Hao-Xuan Bio-Tech Co., Ltd. (Xi'an, China). The human OS cell line 143B was obtained from the American Type Culture Collection (Manassas, VA, USA). Pifithrin-α (PFT-α) was purchased from Selleck Chemicals (Houston, TX, USA). UA and PFT-α were dissolved with dimethyl sulfoxide (DMSO) for experiments in vitro. For in vivo experiments, UA was suspended in 0.4% carboxymethylcellulose sodium. The primary antibodies rabbit anti-human STAT3 and p-STAT3 were obtained from Abcam (Cambridge, MA, USA), and other antibodies were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Cells were cultured with DMEM (containing 10% FBS, 100 U/ml of penicillin and 100 µg/ml of streptomycin). Cells were incubated in 5% CO 2 and 37˚C.
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