Rabbit polyclonal antibody against Sphk1 (HPA022829) and FSCN1 (HPA005723) were purchased from Sigma-Aldrich. Mouse polyclonal antibody against FSCN1 1 (sc-46675) was bought from Santa Cruz Biotechnology, and rabbit monoclonal antibody against SPHK1 (ab109522) was bought from Abcam. Mouse monoclonal anti-β-actin antibody (A5441) was from Sigma-Aldrich, and anti-Ki67 antibody (M7240) was from Dako. Normal rabbit IgG (2729), NFκB p65 (8242), and H3K4me3 (9727) were all from Cell Signaling Technology. p- NFκB p65 (S536) (ab86299) was purchased from Abcam. The in situ cell death detection kit (TUNEL technology, 11684817910) was from Roche. The horseradish peroxidase–linked secondary antibodies against mouse (NA931) and rabbit (NA934) were from GE Healthcare. Actinomycin D (A9415) was from Sigma-Aldrich. A cell permeable peptide (CAS 213546-53-3) that inhibits translocation of the NFkB active complex into the nucleus and its corresponding NFkB control peptide (sc-3060) were brought from Santa Cruz Biotechnology. Safingol (CAS 15639-50-6) was purchased from Cayman Chemical. Bortezomib (CAS 179324-69-7) was purchased from EMD Millipore. CAPTISOL® (20g) was kindly provided by Cydex Pharmeceuticals.
Sc 3060
Manufactured by Santa Cruz Biotechnology
Sourced in United States, United Kingdom
The Sc-3060 is a piece of laboratory equipment designed for general research purposes. It provides a stable and controlled environment for various experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Apocynin apo, by Merck Group (3 mentions)
Neutralizing anti lox antibody, by Abcam (3 mentions)
Jsh 23, by Merck Group (2 mentions)
Sc 3061, by Santa Cruz Biotechnology (2 mentions)
Diphenyleneiodonium dpi, by Merck Group (2 mentions)
Glutathione (gsh), by Merck Group (2 mentions)
Lox 1 inhibitor κ carrageenan, by Merck Group (2 mentions)
N acetylcysteine nac, by Bio-Techne (2 mentions)
Actinomycin d, by Merck Group (1 mentions)
Hpa005723, by Merck Group (1 mentions)
Sc 46675, by Santa Cruz Biotechnology (1 mentions)
Ab109522, by Abcam (1 mentions)
Anti ki67 antibody m7240, by Agilent Technologies (1 mentions)
Fitc conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Pepstatin a, by Merck Group (1 mentions)
Bafilomycin a1, by Merck Group (1 mentions)
Rapamycin, by Merck Group (1 mentions)
E sis3, by MedChemExpress (1 mentions)
Gw 788388, by MedChemExpress (1 mentions)
Fitc dextran 40 kda, by Merck Group (1 mentions)
10 protocols using sc 3060
1
Antibody-Based Profiling of Sphk1 and FSCN1 in Research
2
Visualizing NF-κB Translocation in NKILA Cells
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1 × 103 S26 cells overexpressing NKILA or NKILA shRNA were cultured on coverslips overnight prior to the experiment. After fixing with 4% paraformaldehyde, IF was done and imaged as previously reported [50 (link)], primary antibodies against P65 was used, followed by FITC-conjugated secondary antibodies (Invitrogen, Carlsbad, CA). Sc-3060(Santa Cruz, CA) and JSH-23(Millipore, Billerica, MA) are drugs which inhibit NF-kB nuclear translocation. 30min before specified treatment,10uM Sc-3060 and 5uM JSH-23 were added into the culture.
3
Modulating Autophagy and NF-κB in Invasion Assays
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To inhibit lysosomal protease, 10 μg/mL E-64d (permeable form, Sigma, USA) dissolved in DMSO and 2.5 μg/mL pepstatin A (Sigma) in 10% acetic acid in methanol were used for 4 h. Rapamycin 1 μM (Sigma) was used to induce autophagy in invasion assays. Bafilomycin A1 0.1 μM (Sigma) was used to inhibit autophagy in invasion assays. To inhibit NF-κB activity, an NF-κB inhibitor (100 μg/mL, sc-3060, Santa Cruz, USA), which has been previously used to specifically inhibit NF-kB activity63 (link),64 (link), or the NF-κB control (sc-3061, Santa Cruz) was used.
4
Inhibitory Agents for Cellular Assays
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The following reagents and inhibitors were used: LOX-1 inhibitor κ-carrageenan (250 μg/mL, Sigma-Aldrich), neutralizing anti-LOX antibody (1:50, Abcam, Cambridge, UK), NAD(P)H oxidase inhibitor, diphenyleneiodonium (DPI, 10 μM, Sigma-Aldrich), NAD(P)H oxidase inhibitor, apocynin (Apo, 10 mM, Sigma-Aldrich), a reduced form of glutathione (GSH, 1 mM, Sigma-Aldrich), cell permeable antioxidant N-Acetylcysteine (NAC, 5 mM, Tocris, Bristol, UK), NF-κB inhibitor (SC-3060, 5 μM, Santa Cruz Biotechnology, Dallas, TX, USA), JSH-23 (30 μM, Sigma-Aldrich, USA), ALK5 inhibitor, GW-788388 (5 μg/mL) and SB-431542 (30 μM) (MedChemExpress, Monmouth Junction, NJ, USA), Smad3 inhibitor SIS3 (10 μM) and (E)-SIS3 (5 μM) (MedChemExpress, USA), and FITC-dextran 40 kDa (0.5 mg/mL, Sigma-Aldrich, USA). All the inhibitors were added 1 h before and were maintained throughout the treatment. The buffers and salts were purchased from Merck Biosciences.
5
Inhibition of LOX-1 Signaling Pathways
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The following reagents and inhibitors were used: LOX-1 inhibitor κ-carrageenan (250 μg/ml, Sigma-Aldrich), neutralizing anti-LOX antibody (1:50, Abcam), NAD(P)H oxidase inhibitor, diphenyleneiodonium (DPI, 10 μM, Sigma-Aldrich), NAD(P)H oxidase inhibitor, apocynin (Apo, 10 mM, Sigma-Aldrich), cell permeable antioxidant N-Acetylcysteine (NAC, 5 mM, Tocris), reduced form of glutathione (GSH, 1 mM, Sigma-Aldrich), NF-κB inhibitor (SC3060, 5 μM, Santa Cruz Biotechnology), GATA inhibitor (K-7174, 10 μM, Sigma-Aldrich). PKC inhibitor (bisindolylmaleimide I (BIM), 1 μM, Calbiochem), P38 inhibitor (SB 239063, 10 μM, Sigma-Aldrich), PI3K inhibitor (LY 294002, 1 μM, Sigma-Aldrich), Akt inhibitor (LY 2780301, 10 μM, Sigma-Aldrich), exocytosis inhibitor (TAT-NSF700, 10 μM, Anaspec Inc., CA, USA). All inhibitors were added 1 h before and maintained throughout the treatment. Buffers and salts were purchased from Merck Biosciences.
6
Purification and Analysis of Recombinant CGL
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Natural CGL was isolated as described previously16 (link). Recombinant CGL was prepared according to our previous study19 (link) and further purified using Pierce High Capacity Endotoxin Removal Spin Columns (Thermo Fisher Scientific, USA). LPS (from Escherichia coli 0111:B4), N-acetylcysteine (NAC), PD98059, SP600125, SB203580, PDTC and mouse antibodies against mouse phospho-ERK1/2, phospho-JNK1/2, phospho-p38 and actin were purchased from Sigma-Aldrich (St. Louis, MO). Gö6976, Rottlerin, Wortmannin, LY294002, sc-3060, and antibodies against phospho-PKCα/δ, IL-1β, iNOS, COX-2 and IRAK2, as well as secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). IL-1β, IL-6, TNF-α and MCP-1 ELISA kits were purchased from R&D Systems (Minneapolis, MN). Pierce™ LAL Chromogenic Endotoxin Quantitation Kit was purchased from Thermo Scientific (Rockford, IL).
7
Immortalized Tubular Cell Sepsis Model
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The immortalized human tubular epithelial cell line HK2 (ATCC, Manassas, VA, USA) was commercially available. The immortalized mTECs (mouse kidney proximal tubular epithelial cell line) was a gift from prof. Hui-Yao Lan from The Chinese University of Hong Kong. Cells were cultured in DMEM/F12 (Invitrogen) supplemented with 10% FBS (Invitrogen) at 37 °C in humidified 5% CO2. LPS (10 μg/mL, Sigma Aldrich) was used to stimulate TECs to mimic a cellular sepsis model. For depletion of p65 or circFkbp5, mTECs were transfected with siRNAs targeting p65 mRNA or circFkbp5 (Genepharma, Suzhou, China) with Opti-MEM and Lipofectamine 2000 (Invitrogen) under the guidance of the manufacturer’s instructions. JSH-23 (10 μM, Selleck) or sc-3060 (10 μM, Santa Cruz), two inhibitors to block NF-κB nuclear translocation were separately used to treat cells 12 h before LPS stimulation.
8
NF-kB Inhibition Impacts EPC Migration
9
Benzo(a)pyrene Cytotoxicity in Breast Cancer Cells
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Prior to treatment, cells (MCF-7 and MDA-MB-231) plated at a density of 25,000 cells/well in 24-well plates were supplemented with 5% dextran-coated charcoal-stripped FBS (stripped media) for 72 h. Cells were treated with various concentrations of B(a)P (10− 9–10− 6 M) (Sigma-Aldrich) dissolved in dimethyl sulphoxide (DMSO) to give a concentration of 0.1% DMSO in the final incubate. In some experiments, cells were treated with NFkappaB (NFκB) inhibitor (25 µM, sc-3060) (Santa Cruz Biotechnology Inc) dissolved in RNAase/DNAase-free water. Treatment-induced cytotoxicity was determined by counting cells in a haemocytometer and assessing cell viability by TrypanBlue exclusion (GIBCO, Life technologies).
10
Modulation of Oxidative Stress Pathways
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The following reagents and inhibitors were used: NF-κB inhibitor (SC3060, 5 μM, Santa Cruz Biotechnology), TNF-α receptor inhibitor (R7050, 5 μM, Tocris), LOX-1 inhibitor κ-carrageenan (250 μg/ml, Sigma-Aldrich), neutralizing anti-LOX antibody (1:50, Abcam), NAD(P) H oxidase inhibitor, diphenyleneiodonium (DPI, 10 μM, Sigma-Aldrich), NAD(P)H oxidase inhibitor, apocynin (Apo, 10 mM, Sigma-Aldrich), cell permeable antioxidant N-Acetylcysteine (NAC, 5 mM, Tocris), and the reduced (GSH, 1 mM) and oxidizing (GSSG, 2 mM) form of glutathione (Sigma-Aldrich). All inhibitors were added 1 h before and maintained throughout the treatment. Buffers and salts were purchased from Merck Biosciences.
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