The procedures for all animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh. The procedures were carried out in "accordance" with the approved guidelines. Mice 6–10 weeks old were used. The PUMA+/+ and PUMA−/− littermates on C57BL/6 background (F10)48 (link) and Lgr5-EGFP (Lgr5-EGFP-IRES-creERT2) mice25 (link) have been described. Mice were subjected to whole body irradiation (TBI)11 (link), or abdominal irradiation (ABI) as described30 (link). Mice were injected intraperitoneally (i.p.) with 2 mg/kg of CHIR99021 (Cat# C-6556, LC Laboratories, Woburn, MA) 4 h before radiation or 1 mg/kg of SB415286 (Cat# 1617, Tocris bioscience, Ellisville, MO) 28 h and 4 h before radiation. Mice were sacrificed to collect small intestines for histology analysis and western blotting. All mice were injected i.p. with 100 mg/kg of BrdU (Cat# 858811, Sigma-Aldrich) before sacrifice. Three or more mice were used in each group. More details are found in supplemental material .
Sb415286
Manufactured by Bio-Techne
Sourced in United Kingdom
SB415286 is a lab equipment product manufactured by Bio-Techne. It is a small molecule inhibitor designed for use in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Sb216763, by Bio-Techne (3 mentions)
Pd98059, by Bio-Techne (2 mentions)
Bromodeoxyuridine (brdu), by Merck Group (1 mentions)
Chir99021, by LC Laboratories (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Pi 828, by Bio-Techne (1 mentions)
10 debc, by Bio-Techne (1 mentions)
Pd198306, by Bio-Techne (1 mentions)
Sl327, by Bio-Techne (1 mentions)
Ly294002, by Bio-Techne (1 mentions)
Sb202190, by Bio-Techne (1 mentions)
Tocriscreen kinase inhibitor toolbox, by Bio-Techne (1 mentions)
6 mb camp, by Biolog (1 mentions)
Facscanto, by BD (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
6 bromoindirubin 3 oxime bio, by Cayman Chemical (1 mentions)
Stemspan media, by STEMCELL (1 mentions)
Ar a014418, by Bio-Techne (1 mentions)
Myo inositol, by Merck Group (1 mentions)
Fr180204, by Bio-Techne (1 mentions)
10 protocols using sb415286
1
Investigating Intestinal Radiation Responses
2
Preparation of Amyloid-β Peptide and Kinase Inhibitors
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PDTC (Sigma-Aldrich) was dissolved in sterile water and used immediately after preparation. The kinase inhibitors 10-DEBC, FPA-124, LY294002, PD198306, PD98059, PI828, SB415286, and SL327 (Tocris) were dissolved in DMSO to 10 mM. Aβ(1-42) was purchased from American Peptide (Sunnyvale, CA) and dissolved to 1 mg/mL in sterile water. Protein concentration of the peptide was verified by DC protein assay kit (Bio Rad). The soluble peptide was used for experiments immediately after preparation as described [13 (link)].
3
Expanded Kinase Inhibitor Toolbox
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The Tocriscreen Kinase Inhibitor Toolbox containing 80 kinase inhibitors was supplemented with 10 additional inhibitors (Tocris Bioscience)28 (link). SB415286, SB216763, BIO, H89 and SB202190 were obtained from Tocris Bioscience. 6-MB-cAMP was from Biolog. All compounds were dissolved in DMSO, except for 6-MB-cAMP which was dissolved in water.
4
Screening GSK-3 Inhibitors on BM-MNCs
5
PI3K Activation via Transduction Peptide
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Activation of PI3K was achieved by a transduction peptide (PTD4-PI3KAc) with the following sequence: YARAAARQARAGSDGGpYMDMS [29 (link),46 (link)–48 (link)]. This is a cell membrane permeable phosphopeptide composed of a transduction domain of the tat family [48 (link)] fused to a SH2 interacting domain that allows the activation of the class I PI3K independently from tyrosine kinase dimerization [29 (link),48 (link)–49 (link)]. Peptides were purchased either from GenScript (NJ, USA) or BioPeptide (FR). GSK3 inhibitors: SB-415286 and AR-A014418 [50 (link)–52 (link)] were from Tocris, UK (ref. 1617) and from Sigma, USA (ref. A3230), respectively.
6
Pharmacological Modulation of Cellular Processes
7
Molecular Signaling Pathway Analysis
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Reagents used in this study were obtained from the following sources: Antibodies to Phospho-p70 S6 Kinase (Thr389) (1A5) (Cat# 9206) (1:1000), p70 S6 Kinase (Cat# 9202) (1:1000), 4E-BP1 (Cat# 9644) (1:1000), Phospho-4E-BP1 (Ser65) (Cat# 9456) (1:1000), TFEB (Cat# 4240) (1:1000), phospho-GSK3-beta (Ser9) (D3A4) (Cat# 9322) (1:1000), GSK3-beta (27C10) (Cat# 9315) (1:1000), were from Cell Signaling Technology; antibody to GFP (Cat# ab13970) (1:5000) was from Abcam; antibody to GAPDH (6C5) (Cat# sc-32233) (1:20000) was from Santa Cruz; antibody to TFEB-pSer142 (Cat# ABE1971) (1:15000) was from EMD-Millipore; antibodies to ERK1/ERK2 (Cat# MAB1576) (1:1000) and phospho-ERK1 (T202/204)/ERK2 (T185/Y187) (Cat# AF1018) (1:1000) were from R&D Systems; antibodies to TFEB-pS138 (1:15000) were custom generated in collaboration with Bethyl Laboratories. Both TFEB-pSer142 and TFEB-pS138 antibodies tested in this study showed marginal cross-reactivity with unphosphorylated TFEB (Fig. 4c ).
Chemicals: Torin 1 (Cat# 4247) was from Tocris; Leptomycin B (Cat# L2913), Protease Inhibitor Cocktail (Cat# P8340) and puromycin (Cat# P9620) were from Sigma-Aldrich; U0126 (Cat# 9903) was from Cell Signaling; SB415286 (Cat# 1617) was from TOCRIS; PhosSTOP phosphatase inhibitor cocktail tablets (Cat# 04906837001) were from Roche.
Chemicals: Torin 1 (Cat# 4247) was from Tocris; Leptomycin B (Cat# L2913), Protease Inhibitor Cocktail (Cat# P8340) and puromycin (Cat# P9620) were from Sigma-Aldrich; U0126 (Cat# 9903) was from Cell Signaling; SB415286 (Cat# 1617) was from TOCRIS; PhosSTOP phosphatase inhibitor cocktail tablets (Cat# 04906837001) were from Roche.
8
Estrogen-Dependent Mechanical Loading in Mice
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When aged 12 weeks, mice underwent bilateral OVX or Sham operation (non-OVX) by dorsal midline incision under 2% isoflurane inhalation anesthesia. Analgesia was ensured by oral Tramal administration (25 mg/L drinking water) until day 3 post surgery. To avoid wound infection, the mice received 45 µg/g clindamycin-2-dihydrogenphosphate by subcutaneous injection directly before surgery. At 4 weeks after surgery and 2 days before mechanical loading was applied, a subcutaneous estradiol pellet (0.18 mg 17β-estradiol per 60-day release pellet (Innovative Research of America, Sarasota, FL, USA) or a placebo pellet was applied subcutaneously in the neck (Scheme 1 ). At 6 weeks after surgery, blood samples were collected for serum estradiol analysis by ELISA (Estradiol ELISA, Calbiotech, El Cajon, CA, USA), and uteri were removed and weighed.
To activate Wnt/β-catenin signaling, 1 mg/kg body weight of the GSK-3β inhibitor SB415286 (3-(3-chloro-4-hydroxyphenylamino)-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione, Tocris Bioscience, Bristol, UK) was subcutaneously injected daily during the time period of mechanical loading. Control mice received vehicle solution.
To activate Wnt/β-catenin signaling, 1 mg/kg body weight of the GSK-3β inhibitor SB415286 (3-(3-chloro-4-hydroxyphenylamino)-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione, Tocris Bioscience, Bristol, UK) was subcutaneously injected daily during the time period of mechanical loading. Control mice received vehicle solution.
9
Murine Dendritic Cell Profiling
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Recombinant mouse (rm) granulocyte macrophage colony- stimulating factor (GM-CSF), rm IL-4, and rm IFN-γ were purchased from R&D Systems (Minneapolis, MN, USA). Resveratrol (＞99% purity) was obtained from Sigma-Aldrich (St. Louis, MO, USA). SB415286, a GSK-3 inhibitor, was obtained from Tocris Bioscience (Bristol, UK). Fluorescein isothiocyanate (FITC)- and phycoerythrin (PE)-conjugated monoclonal antibodies (Abs) used to detect expression of CD11c (HL3), CD4 (L3T4), and CD8 (Lyt-2) were purchased from BD Pharmingen (San Diego, CA, USA). To detect protein levels by Western blotting, anti-phosphoserine-GSK-3β (Ser9) was purchased from Cell Signaling Technology (Beverly, MA, USA). Polyclonal rabbit anti-mouse Abs against TTS and α-tubulin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).
10
Cardioprotective Strategies in Ischemia-Reperfusion
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All hearts were subjected to 20 min of stabilization followed by 30 min of regional ischemia (RI) and 120 min of reperfusion (Fig. 1). Hearts were randomized to receive either vehicle (Ctr), insulin (Ins [0.3 mU/ml]; Novo Nordisk A/S, Bagsvaerd, Denmark) or the GSK3β inhibitor SB415286 (SB41 [3μM]; Tocris Bioscience, UK) for 15 min at the onset of reperfusion, with or without interruption by 3x30s global ischemia (IPost). In addition, the combination of insulin and GSK3β inhibition for 15 min at reperfusion was evaluated. Finally, a parallel set of hearts underwent the same protocols, but were harvested at 15 min reperfusion. The atria and right ventricle were removed and the area at risk from the left ventricle was snap frozen in liquid nitrogen for protein determination by western blotting (WB, n=3-5 for each group).
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