A549 cells were treated with calf Intestine alkaline phosphatase (CIAP, New England Biolabs) and incubated at 37°C for 30 min before being lysed in SDS loading buffer and electrophoresis. ZAP-S was detected by WB using ZC3HAVI antibody (Proteintech) and mobility compared with an untreated control. For investigation of effects of kinase inhibitors on ZAP phosphorylation, A549 cells were treated with 2 μM C16 (Sigma), 5 μM GSK3β inhibitor SB216763 (Sigma) or DMSO for 3 h. Cells were washed with PBS before being lysed in SDS loading buffer. To investigate effects of phosphorylation inhibitors on virus replication, A549 cells were similarly pre-treated with 2 μM C16, 5 μM SB216763 or DMSO for 3 h, prior to infection at an MOI of 10. Supernatant harvested at 24 h was assayed for infectivity in A549 cells as described above.
Gsk3β inhibitor sb216763
Manufactured by Merck Group
Sourced in United States
SB216763 is a selective inhibitor of the enzyme glycogen synthase kinase-3 beta (GSK3β). GSK3β is involved in various cellular processes and is a key regulator of numerous signaling pathways. SB216763 binds to and inhibits the activity of GSK3β, which may be used in research applications to investigate the role of GSK3β in different biological systems.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Cell Signaling Technology (2 mentions)
E cadherin, by Cell Signaling Technology (2 mentions)
β catenin, by Cell Signaling Technology (2 mentions)
Gsk3β, by Cell Signaling Technology (2 mentions)
N cadherin, by Cell Signaling Technology (2 mentions)
Vimentin, by Cell Signaling Technology (2 mentions)
Dimethyl sulfoxide (dmso), by Solarbio (1 mentions)
Emodin, by Merck Group (1 mentions)
Phosphorylated akt p akt, by Cell Signaling Technology (1 mentions)
Claudin 1, by Cell Signaling Technology (1 mentions)
Rs504393, by Bio-Techne (1 mentions)
Tak 242, by Merck Group (1 mentions)
Bindarit, by Cayman Chemical (1 mentions)
Anti ccr 2 antibody, by Abcam (1 mentions)
Salinomycin, by Merck Group (1 mentions)
β catenin, by Santa Cruz Biotechnology (1 mentions)
Mg132, by Merck Group (1 mentions)
Akt inhibitor, by Merck Group (1 mentions)
Lps escherichia coli 0111 b4, by Merck Group (1 mentions)
5 protocols using gsk3β inhibitor sb216763
1
Phosphorylation Regulation of ZAP-S and Virus Replication
2
Emodin and GSK-3β Inhibitor Protocol
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Emodin and GSK-3β inhibitor (SB216763) were purchased from Sigma Aldrich (St. Louis, MO, USA). Dimethylsulfoxide (DMSO) was obtained from Solarbio (Beijing, China). The Emodin or SB216763 was diluted in corresponding medium, and the DMSO's final concentration was under 0.1% (v/v). The antibodies E-cadherin, N-cadherin, Vimentin, Claudin-1, Slug, ILK, Akt, phosphorylated Akt (p-Akt), GSK-3β, β-catenin, and GAPDH were purchased from Cell Signaling Technology (MA, USA). And phosphorylated GSK-3β (p-GSK-3β) was obtained from Santa Cruz Biotechnology (CA, USA).
3
Inhibiting Inflammatory Pathways in Research
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Bindarit, an inhibitor of MCP-1 synthesis, was purchased from Cayman chemical (Ann Arbor, MI, USA). CCR2 antagonist RS504393 was purchased from TOCRIS, Inc. (Minneapolis, MN, USA). TLR4 inhibitor TAK242 was purchased from EMD Millipore, Inc. (Burlington, MA, USA). GSK3β inhibitor SB-216763 was purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Anti-MCP-1 antibody was purchased from Bio-Rad AbD Serotec, Inc. (Raleigh, NC, USA). Anti-CCR-2 antibody was purchased from BioVision, Inc. (Milpitas, CA, USA). Anti-Iba-1 antibody was purchased from Wako Chemicals USA, Inc. (Richmond, VA, USA). All other antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Other chemicals/reagents used in this project were obtained from Thermo Fisher Scientific, Inc. (Waltham, MA, USA) unless stated otherwise.
4
Salinomycin and GSK-3β Inhibitor Effects
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Salinomycin and GSK-3β inhibitor (SB216763) were purchased from Sigma Aldrich (St Louis, MO, USA). Respective stock solutions (400 μM and 50 nM) were prepared with dimethyl sulfoxide (DMSO) and stored at −20°C. The working concentration was diluted in the corresponding medium, and the DMSO’s final concentration was under 0.1% (v/v). Control groups were treated with an equal volume of DMSO. The antibodies E-cadherin, N-cadherin, vimentin, keratin, GSK-3β, β-catenin and GAPDH (Cell Signaling Technology, MA, USA), phosphorylated GSK-3β-ser9 (p-GSK-3β-ser9; Santa Cruz Biotechnology, TX, USA) and Slug (Abcam, Cambridge, UK) were used for Western blot assay. The antibody β-catenin (Santa Cruz) was used for immunofluorescence assay.
5
Investigating TAK1 Signaling Pathway Modulation
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PAR-1-activating peptide (TFLLRNPNDK-NH2) was custom synthesized as the C-terminal amide with a purity of >95% by Genscript (Piscataway, NJ). Scrambled-siRNA (Sc-siRNA) and human (h)-specific siRNA to target TAK1, sequences 5′-GUGCUGACAUGUCUGAAAUTT-3′; 5′-AUUUCAGACAUGUCAGCAC-3′, were custom synthesized by IDT (Coralville, IA). LPS Escherichia coli 0111:B4 (Cat# L3012), TAK1 inhibitor OZ (Cat# 09890), MG-132 (Cat# 474790), GSK3β inhibitor SB216763 (Cat# S3442), and AKT inhibitor (Cat# A6730) were from Sigma (St. Louis, MO). CaMKKβ inhibitor 7-Oxo-7H-benzimidazo [2,1-a]benz[de]isoquinoline-3-carboxylic acid acetate (STO-609 acetate; Cat# 1551) was from Tocris Bioscience. TAK1 expression constructs (HA-WT-TAK1 and HA-TAK1K63W kinase-defective mutant) were a gift from J. Ninomiya-Tsuji (North Carolina State University, Raleigh, NC). In situ cell death detection (TUNEL Assay) kit, TMR red (Cat# 12156792910) was obtained from Roche Diagnostic.
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