Gsk650394

Manufactured by Bio-Techne

Sourced in United Kingdom, United States

GSK650394 is a laboratory reagent product manufactured by Bio-Techne. It is a small molecule inhibitor that targets the SGK1 (serum and glucocorticoid-regulated kinase 1) protein.

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Lab products found in correlation

22 protocols using gsk650394

Calcium Signaling in Cell Superfusion

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Cells were superfused normally with a solution containing (mM): 88 NaCl, 5 KCl, 5.5 glucose, 1 CaCl₂, 10 HEPES and 100 mannitol, adjusted to pH 7.4 with NaOH (300 mosm kg⁻¹ H₂O). The HTS was adjusted to 200 mosm Kg⁻¹ H₂O by omitting mannitol. 4-αPDD was acquired from Sigma (St Louis, MO, USA). Fluo-4AM and Fura-2AM were acquired from Molecular Probes, Inc (Eugene, OR, UK). Stock solutions of phorbol esters were initially prepared in dimethyl sulphoxide (DMSO) at a concentration of 1 mM, and then stored at –20 °C. The final DMSO concentration in the experimental bath solution containing phorbol esters never exceeded 0.5%. Insulin and SGK1 inhibitors GSK 650394 were acquired from Tocris Bioscience (Ellisville, MO, USA), and used them as the manufacturer's recommendation.

Shin S.H., Lee E.J., Chun J., Hyun S, & Kang S.S. (2015). Phosphorylation on TRPV4 Serine 824 Regulates Interaction with STIM1. The Open Biochemistry Journal, 9, 24-33.

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Drug Screening for Anti-inflammatory Compounds

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Drug screening was performed on the Spectrum Collection (2010 version; MicroSource Discovery Systems). All compounds were dissolved in DMSO, such that 1% DMSO was used as a negative control (Sigma- Aldrich). Reagents used as positive controls were an inhibitor of SGK, GSK650394 (Tocris Bioscience), at 10 µM and the c-Jun N-terminal kinase inhibitor SP600125 (Sigma-Aldrich) at 30 µM. The panel of known anti-inflammatory compounds was obtained from GlaxoSmithKline. Tanshinone IIA and cryptotanshinone were obtained from Sigma-Aldrich, DMOG was from Enzo Life Sciences, and GM-CSF was from PeproTech.

Robertson A.L., Holmes G.R., Bojarczuk A.N., Burgon J., Loynes C.A., Chimen M., Sawtell A.K., Hamza B., Willson J., Walmsley S.R., Anderson S.R., Coles M.C., Farrow S.N., Solari R., Jones S., Prince L.R., Irimia D., Rainger G.E., Kadirkamanathan V., Whyte M.K, & Renshaw S.A. (2014). A Zebrafish Compound Screen Reveals Modulation of Neutrophil Reverse Migration as an Anti-Inflammatory Mechanism. Science translational medicine, 6(225), 225ra29.

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Cytokine-induced mDCT15 cell signaling

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Cytokines IL-17A, IL-17F, and TNFα were obtained from R&D systems. The SGK1 inhibitor, GSK 650394, was obtained from TOCRIS Bioscience. Mouse distal convoluted tubule (mDCT15) cells were cultured as previously described^{14 (link)}. NEDD4-2 deficient mDCT15 cells were generated as previously described^{15 (link)} HK-2 cells were purchased from ATCC.

Norlander A.E., Saleh M.A., Kamat N.V., Ko B., Gnecco J., Zhu L., Dale B.L., Iwakura Y., Hoover R.S., McDonough A.A, & Madhur M.S. (2016). INTERLEUKIN 17A REGULATES RENAL SODIUM TRANSPORTERS AND RENAL INJURY IN ANGIOTENSIN II-INDUCED HYPERTENSION. Hypertension, 68(1), 167-174.

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Prostate Cancer Cell Line Treatments

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LAPC4 and CWR-22Rv1 cells lines were plated at 5×10⁴ in 6-cm cell culture dishes using standard media listed above, supplemented with either 10 or 1 % charcoal stripped serum as specified. Cells were treated for 2 h, 3 h, 7 h, or 14 days with combinations of the following chemical treatments (or vehicle control) as described below: 1 nM R1881 (Sigma-Aldrich), 10 μM MDV (Selleck Chemicals and Medivation), 100 nM dexamethasone (Sigma-Aldrich), 10 μM Casodex (Sigma-Aldrich), 100 nM mifepristone (Sigma-Aldrich), 1 μM CORT 122928 (Corcept Therapeutics), and 1 μM GSK 650394 (Tocris Bioscience). For doxycycline treatment, cells were grown in 1 μg/ml (in water) doxycycline (Sigma-Aldrich).

Isikbay M., Otto K., Kregel S., Kach J., Cai Y., Vander Griend D.J., Conzen S.D, & Szmulewitz R.Z. (2014). Glucocorticoid Receptor Activity Contributes to Resistance to Androgen-Targeted Therapy in Prostate Cancer. Hormones & Cancer, 5(2), 72-89.

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Dendritic Cell Differentiation and Activation

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Bone-marrow derived dendritic cells (BMDCs) from C57BL/6 mice were cultured from bone marrow in ultra-low binding polystyrene plates with 10ng/ml IL-4 and G-CSF. Cells were preconditioned at least 16 hours prior to use either in normal salt media (DMEM+10% FBS +0mM NaCl, NSM) or high salt media (same +40mM NaCl, HSM). Cells were pre-treated for 30 minutes in the indicated doses of inhibitor, then subjected to 1ng/ml LPS (E. coli, Sigma, Cat# L3024). Supernatants were then collected and analyzed by RnD DuoSet ELISAs. SGK1 inhibitor GSK 650394 (Tocris Biotechne); p38 MAPK inhibitor SB203580 (Invivogen)

Tubbs A.L., Liu B., Rogers T.D., Sartor R.B, & Miao E.A. (2017). Dietary Salt Exacerbates Experimental Colitis. Journal of immunology (Baltimore, Md. : 1950), 199(3), 1051-1059.

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Protein Expression Profiling in Cells

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Cell Signaling (Beverly, MA) antibodies: EGF receptor variant III (EGFRvIII) (Cat# 64952), p-Akt (S473; Cat# 4060), p-NDRG1 (T346; Cat# 5482), Rictor (Cat# 2114), 5-methylcytosine (5-mC) (Cat# 28692), DNMT3A (Cat #3598), acetylated-lysine (Cat# 9441), H3 p.K27me2 (Cat# 9755), H3 p.K27me3 (Cat# 9733), Histone H3 (Cat# 4499), EZH2 (Cat# 5246), FAK (Cat# 13009), p-FAK (Y397; Cat# 8556), β-actin (Cat# 3700), GAPDH (Cat# 5174), HRP-linked anti-rabbit IgG (Cat# 7074) and HRP-linked anti-mouse IgG (Cat# 7076). Santa Cruz (Dallas, TX) antibodies: p-PKC α (S657; Cat# sc-377565). GeneTex (Irvine, CA) antibodies: 5-mC (Cat# GT4111). Thermo Fisher antibodies: GRIA1 (Cat# PA5-95207). DAKO (Glostrup, Denmark) antibodies: Synaptophysin (Cat# M731529). Millipore (Burlington, MA) antibodies: Nestin (Cat# MAB5326).
Reagents used are sodium acetate (Sigma; Cat # S5636), Trichostatin A (TSA) (Sigma; Cat# T1952), PP242 (Cayman Chemical, Ann Arbor, MI; Cat# 13643), Akti-1/2 (Calbiochem, La Jolla, CA; Cat# 124018), Bisindolylmaleimide I (Bis-I) (Santa Cruz; Cat# sc-24003), GSK 650394 (Tocris Bioscience, Bristol, UK; Cat# 3572/10), GSKJ4 (Sigma; Cat# T1952), GSK126 (MedChem Express, Monmouth Junction, NJ; Cat# HY-13470), GSK2256098 (Selleck Biotech, Kanagawa, Japan; Cat# S8523) and Philanthotoxin-7,4 (PhTx-74) (Abcam, Cambridge, UK; Cat# ab120257).

Harachi M., Masui K., Shimizu E., Murakami K., Onizuka H., Muragaki Y., Kawamata T., Nakayama H., Miyata M., Komori T., Cavenee W.K., Mischel P.S., Kurata A, & Shibata N. (2024). DNA hypomethylator phenotype reprograms glutamatergic network in receptor tyrosine kinase gene-mutated glioblastoma. Acta Neuropathologica Communications, 12, 40.

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Inflammatory Signaling Pathway Modulators

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LPS from Escherichia coli 0111:B4, oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), rotenone, mitomycin C, HMGB1, and BrdU were purchased from Sigma-Aldrich. The following kinase inhibitors were purchased from Tocris: leucine-rich repeat kinase 2-IN-1, BMS509744, GSK650394, and GSK2250665A. IRAK-1/4 inhibitor 1 was purchased from Sigma-Aldrich, SBI-0206965 and GNF-2 were obtained from Selleckchem, P-M2tide was obtained from Enzo Life Sciences, and MlkL inhibitor was obtained from Calbiochem. AZD7545 was kindly provided by Professor In-Kyu Lee at Kyungpook National University.

Kim J.H., Han J., Afridi R., Kim J.H., Rahman M.H., Park D.H., Lee W.S., Song G.J, & Suk K. (2023). A multiplexed siRNA screen identifies key kinase signaling networks of brain glia. Life Science Alliance, 6(5), e202201605.

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Antibodies and Reagents for CFTR Localization

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The antibodies used were: rabbit anti-SGK1 antibody (Sigma); mouse anti-CFTR C-terminus antibody (clone 24-1; R&D systems, Minneapolis, MN); mouse anti-CFTR antibody (clone 596; University of North Carolina Cystic Fibrosis Center, Chapel Hill, NC); mouse anti-ezrin, mouse anti-Rab5a and mouse anti-EEA1 antibodies (BD Biosciences, San Jose, CA); rabbit anti-Rab 11a (Life Technologies Corp); mouse anti-EGFR (Santa Cruz Biotechnology, Inc, Santa Cruz, CA); horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit secondary antibodies (Bio-Rad, Hercules, CA). SGK1-S442D, a constitutively active form of human SGK1 and human SGK1-K127N, an inactive form of human SGK1, were constructed as described [15] . The SGK inhibitor GSK 650394 was obtained from Tocris (purchased through R&D Systems, Minneapolis, MN). All antibodies and reagents were used at the concentrations recommended by the manufacturers.

Bomberger J.M., Coutermarsh B.A., Barnaby R.L., Sato J.D., Chapline M.C, & Stanton B.A. (2014). Serum and Glucocorticoid-Inducible Kinase1 Increases Plasma Membrane wt-CFTR in Human Airway Epithelial Cells by Inhibiting Its Endocytic Retrieval. PLoS ONE, 9(2), e89599.

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PTOV1 Protein Degradation Mechanisms

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Lysosomal and proteasomal degradation experiments utilized MG132 (Selleckchem, Houston, TX, USA cat. S2619) and Bafilomycin (Cayman Chemical Company, Ann Arbor, Michigan, USA cat. 11038). PC3 cells stably expressing FLAG-PTOV1 constructs or HEK-293T cells transiently transfected with GFP-PTOV1 constructs were treated (2 days after transfection) with either 10 μM MG132, 100 nM Bafilomycin, or a similar volume of vehicle control (DMSO) for 2 hours prior to harvest.
PTOV1 degradation rates were assessed using cycloheximide (CHX, Cayman Chemical Company, cat 14126). PC3 cells stably expressing FLAG-PTOV1 WT or S36A were treated with 50 μg/ml CHX or an equal volume of vehicle control (DMSO). Alternatively, HEK-293T cells were transiently transfected with FLAG-PTOV1 S36A or FLAG-NES-PTOV1 S36A and treated with 50 μg/ml CHX two days post-transfection. Cells were harvested at the indicated timepoints and lysed in RIPA lysis buffer.
SGK2 inhibition experiments were performed using GSK 650394 (Tocris, Minneapolis, MN, USA cat. 3572). Cells were treated with 10 μM GSK 650394 for 48 hours prior to harvest.

Pennington K.L., McEwan C.M., Woods J., Muir C., Sahankumari A.G., Eastmond R., Balasooriya E.R., Egbert C.M., Kaur S., Heaton T., McCormack K.K., Piccolo S.R., Kurokawa M, & Andersen J.L. (2021). SGK2, 14-3-3 and HUWE1 cooperate to control the localization, stability and function of the oncoprotein PTOV1. Molecular cancer research : MCR, 20(2), 231-243.

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PC12 Cell Culture and SGK1 Inhibition

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The rat adrenal phaeochromocytoma PC12 cell line was purchased from the Bioresource Collection and Research Center, Hsinchu, Taiwan (BCRC#60048). PC12 cells were maintained in DMEM media using 10% horse serum, 5% fetal bovine serum, 4.5 g/mL glucose, penicillin, and streptomycin. Prior to the experiments, the PC12 cell line was cultured until it reached 60–80% confluency on culture dishes coated with collagen type IV (Sigma-Aldrich, St. Louis, MO, USA; #C5533). Additionally, our research did not involve the differentiation of PC12 cells [18 (link),19 (link)]. For SGK1 inhibition, the SGK1 inhibitor GSK650394 (Tocris Bioscience, Bristol, UK; #890842-28-1) was used. Following overnight incubation, the medium was replaced with a growth medium containing GSK650394 (100 μM) for the indicated times. The mRNA expression and protein levels of SGK1 and selected DEGs were analyzed using real-time PCR and Western blotting.

Li Y.H., Sun C.C., Chen P.M, & Chen H.H. (2023). SGK1 Target Genes Involved in Heart and Blood Vessel Functions in PC12 Cells. Cells, 12(12), 1641.

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