Carbonyl cyanide m-chlorophenyl hydrazine (CCCP) and okadaic acid were from Sigma (Sigma-Aldrich, St. Louis, MO, USA). AZD1080, CHIR98014, cyclosporin A, GNE7915, LRRK2in1, oligomycin, rotenone, SB216763, sorafenib, staurosporine, and valinomycin were from Cayman Chemical Company (Ann Arbor, MI, USA). Tipranavir (#11285) was obtained through the NIH’s HIV Reagent Program, which is supported by National Institute of Allergy and Infectious Diseases. 10 mM stock solutions in dimethyl sulfoxide (DMSO) were prepared for all compounds with the exception of staurosporine (1mg/ml in ethyl acetate) and stored at −20°C until further use. Working dilutions were prepared fresh for each experiment.
Sb216763
Manufactured by Cayman Chemical
Sourced in United States
SB216763 is a small molecule that inhibits the enzyme GSK3β (Glycogen Synthase Kinase 3 beta). It is a white, crystalline powder that is soluble in DMSO and other organic solvents. SB216763 is commonly used as a research tool to study the role of GSK3β in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Ly294002, by Cayman Chemical (2 mentions)
Aminopterin, by Cayman Chemical (2 mentions)
Vitamin e α tocopherol acetate, by Cayman Chemical (2 mentions)
Nicotinamide, by Cayman Chemical (2 mentions)
Ci994, by Cayman Chemical (2 mentions)
Sb 415286, by Cayman Chemical (2 mentions)
Chir99021, by Cayman Chemical (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Lithium chloride (licl), by Merck Group (2 mentions)
Oligomycin, by Cayman Chemical (1 mentions)
Rotenone, by Cayman Chemical (1 mentions)
Valinomycin, by Cayman Chemical (1 mentions)
Staurosporine, by Cayman Chemical (1 mentions)
Sorafenib, by Cayman Chemical (1 mentions)
Carbonyl cyanide m chlorophenyl hydrazine cccp, by Merck Group (1 mentions)
Azd1080, by Cayman Chemical (1 mentions)
Cyclosporin a, by Cayman Chemical (1 mentions)
Lrrk2in1, by Cayman Chemical (1 mentions)
Okadaic acid, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
10 protocols using sb216763
1
Compound Screening for Drug Discovery
2
Keratinocyte Culture Protocol with Cytokines
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Dispase, defined keratinocyte serum-free medium (KSFM) supplemented with keratinocyte growth factor (KGF), high glucose Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Life Technologies (Gibco and Invitrogen, Auckland, CA, USA). SB-216763 and Y27632 were from Cayman Chemicals (Ann Arbor, MI, USA). IL-8, IL-13, IL-17A, TGF-β1and TNF-α were purchased from PeproTech (Rocky Hill, NJ, USA). 4′,6-diamidino-2-phenylindole (DAPI) was purchased from Sigma-Aldrich (St Louis, MO, USA). Fluorescein isothiocyanate (FITC)-conjugated, Cy3-conjugated and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Cocktail protease inhibitors were purchased from Roche Diagnostics (Indianapolis, IN, USA). Primary antibodies used are listed in electronic supplementary material, table S1.
3
Investigating Notch Signaling in Cancer Cells
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CdCl2 was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). LY294002 and SB216763 were obtained from Cayman Chemical Company (Ann Arbor, MI, USA). AG1478 and PPP were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). DAPT and pifithrin-α were obtained from Wako. MK-2206 was obtained from Active Biochem (Maplewood, NJ, USA). Antibodies against phospho-EGFR (Tyr1068), total EGFR (D38B1) XP, phospho-Akt (Thr308) (C31E5E), total Akt (pan) (C67E7), phospho-p70 S6 kinase (Thr389) (108D2), phospho-GSK-3α/β (Ser21/9) (37F11), total GSK3-α/β (D75D3) XP, cleaved Notch1 (Val1744) (D3B8), Notch1 (D1E11) XP, Jagged1 (28H8), Jagged2 (C23D2), Snail (C15D3), E-cadherin (24E10), phospho-p53 (Ser15), and MEK1/2 were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). p53 (DO-1), Notch1 (C-20), actin (I-19), and lamin A/C (14/LaminAC) antibodies were obtained from Santa Cruz Biotechnology. The siRNAs targeted against the human Notch1 (siRNA-1: Hs_NOTCH1_3 FlexiTube siRNA, SI00119028, siRNA-2: Hs_NOTCH1_4 FlexiTube siRNA, SI00119035), Jagged1 (Hs_JAG1_5 FlexiTube siRNA, SI02780134), Jagged2 (Hs_JAG2_2 FlexiTube siRNA, SI03095764), SNAI1 (siRNA-1: Hs_SNAI1_1 FlexiTube siRNA, SI00083398, siRNA-2: Hs_SNAI1_5 FlexiTube siRNA, SI02636424), and non-target siRNA (AllStars Negative Control siRNA) were purchased from Qiagen (Hilden, Germany).
4
Canonical Wnt Signaling and Macrophage Activation
5
Inflammatory Mediators Experimental Protocol
6
Characterization of RCC Cell Lines
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The RCC cell lines ACHN, Caki1, and A498 were obtained from American Type Culture Collection (Manassas, VA, USA). ACHN is derived from pleural effusion in metastatic RCC having wild type of VHL [23 (link), 24 (link)]. Caki1 and A498 cells come from clear cell RCC with VHL wild type [23 (link), 25 (link)], and clear cell RCC with VHL mutation (426_429delTGAC) [25 (link)], respectively. Cells were cultured in RPMI medium supplemented with 50 μg/mL of kanamycin and 10 % fetal bovine serum in an incubator at 5 % CO2 and 37 °C. Human renal proximal tubular epithelial cell (HRPTEpC) was obtained from Cell applications Inc (San Diego, CA, USA). Cells were cultured in RenaEpi cell growth medium with growth supplements in an incubator at 5 % CO2 and 37 °C. AR-A014418 was purchased from Calbiochem (San Diego, CA, USA). Two other GSK-3 inhibitors, SB-216763 and TDZD8, were obtained from Cayman Chemicals (Ann Arbor, MI, USA) and Sigma-Aldrich Japan (Tokyo, Japan), respectively. Rapamycin and everolimus were obtained from Selleck Chemicals (Houston, TX, USA), LY294002 was from Wako Pure Chemical Industries (Tokyo, Japan), recombinant GSK-3β was purchased from New England Biolabs (NEB) Japan (Tokyo, Japan), and recombinant GST-4EBP1 was obtained from Sigma-Aldrich Japan.
7
Melanogenesis Regulation in Keratinocytes and Melanoma Cells
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HaCaT human keratinocytes were kindly provided by Dr. Louis-kuoping Chao. B16F10 mouse melanoma cells were purchased from the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan). Both cell lines were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) (GIBCO, Grand Island, NY, USA) with 10% fetal bovine serum (Thermo Fisher Scientific, Carlsbad, CA, USA) at 37°C in a humidified incubator containing 5% CO2. Calycosin was synthesized by Small Molecule Medicinal Chemistry Laboratory in China Medical University. L-DOPA, Arbutin, and α-MSH were obtained from Sigma-Aldrich (St. Louis, MO, USA); H-89, PD98059, LY294002, SB203580, SB216763, and Propylthiouracil (PTU) were obtained from Cayman Chemical (Ann Arbor, MI, USA). Antibodies used in this study and their sources were as follows: β-actin, MC1R, MITF, Tyrosinase, TRP-1, TRP-2, p38, p-p38, β-Catenin (Santa Cruz Biotechnology, Santa Cruz, CA, USA); p-H2AX Ser139 (Millipore, Billerica, MA, USA); JNK, p-JNK, CREB, p-CREB, ERK, p-ERK, Akt, p-Akt Ser473, GSK3β, p-GSK3β Ser9 (Cell Signaling, Beverly, MA, USA).
8
SOSR-CO Pharmacological Modulation
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Immediately after plating in 96-well plates, the SOSR-COs were treated with various drugs for 48 h. Along with VPA (Cayman Chemical), the SOSR-COs were treated with different HDAC inhibitors, GSK3β inhibitors, folic acid (FA) inhibition or supplementation, and antioxidants. We tested HDAC inhibitors that target different classes: Trichostatin A (Cayman Chemical), Nicotinamide (Cayman Chemical), and CI-994 (Cayman Chemical). The GSK3β inhibitors included CHIR99021 (Cayman Chemical), BIO (Cayman Chemical), SB-216763 (Cayman Chemical), SB-415286 (Cayman Chemical), and lithium chloride (Sigma-Aldrich, St. Louis, MO, USA). FA (Cayman Chemical) was supplemented, and the FA pathway was inhibited by aminopterin (Cayman Chemical). The antioxidant was vitamin E (±-α-Tocopherol Acetate; Cayman Chemical). Appropriate amounts of each treatment were mixed with 3N media without vitamin A, plus the 4 inhibitors. A total of 100 μL of media mixed with the treatment was added per well.
9
Modulation of SOSR cell fate
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Immediately after plating in 96-well plates, SOSRS were treated with various drugs for 48 hours. Along with VPA (Cayman Chemical, 13033), SOSRS were treated with different HDAC inhibitors, GSK3β inhibitors, folic acid (FA) inhibition or supplementation, and antioxidants. We tested HDAC inhibitors that target different classes: Trichostatin A (TSA; Cayman Chemical, 89730), Nicotinamide (Cayman Chemical, 11127), CI-994 (Cayman Chemical, 12084). GSK3β inhibitors included CHIR99021 (Cayman Chemical, 13122), Bio (Cayman Chemical, 13123), SB-216763 (Cayman Chemical, 10010246), SB-415286 (Cayman Chemical, 10010247), and lithium chloride. FA (Cayman Chemical, 20515) was supplemented, and the FA pathway was inhibited by Aminopterin (Cayman Chemical, 21802). Antioxidants included Vitamin E (±-α-Tocopherol Acetate; Cayman Chemical, 28399), and Resveratrol (trans-Resveratrol [RV]; Cayman Chemical, 70675). Appropriate amounts of each treatment were mixed with 3N media without vitamin A plus the 4 inhibitors. 100μL of media mixed with the treatment was added per well. SOSRS were treated for 48 hours before being fixed.
10
Hypoxic Exposure with Signaling Inhibitors
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For hypoxic exposure cells were put in a purpose--built hypoxic glove--box chamber (in vivo 400, RUSKINN Technologies, Guiseley, UK) for different time intervals (37°C, 5% CO2). The oxygen level was maintained at 1%. Inhibitors of Rac1 (Millipore, Billerica, MA, USA) and RhoA (cytoskeleton, Denver, CO, USA) were added just prior to hypoxic exposure. Inhibitors of GSK3β (SB216763, Cayman chemicals, Ann Arbor, MI, USA; LiCl, Sigma) were added 24h before and JNK inhibitor SP600123 was added 1h before cells were subjected to hypoxia. Control cells were incubated under normal conditions (37°C, 21% O2, 5% CO2).
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