Prior to use in cell culture, lovastatin (Calbiochem, Billerica, MA) was chemically activated by alkaline hydrolysis as we have previously described (Von Zee et al., 2009 (link)). Transformed human TM cells were cultured to confluency and treated x 24h with vehicle (0.01% ethanol) or activated lovastatin (10 μM). To inhibit post-translational isoprenylation and functional activation of small monomeric GTPases, primary or transformed human TM cells were incubated x 24h in the absence (0.6% DMSO) or presence of selective inhibitors of farnesyl transferase (FTI-277, 20 μM) or geranylgeranyl transferase-I (GGTI-298, 20 μM; Calbiochem). To inhibit de novo mRNA synthesis, transformed human TM cells were pre-treated x 1h with actinomycin D (1 μg/ml; Sigma-Aldrich) prior to incubation with GGTI-298 (20 μM). To determine the role of specific Rho subfamily GTPases in facilitating constitutive TGF-β2 expression, GTM3 cells were treated x 24h with specific inhibitors of Rac1 (NSC23766, 20 μM; Calbiochem), Cdc42 (ML141, 20 μM; Calbiochem), RhoA/B/C (C3 exoenzyme, 10 μg/ml; Cytoskeleton, Denver, CO), or p160ROCK (Y-27632, 10 μM; Tocris Biosciences, Minneapolis, MN).
Ggti 298
Manufactured by Merck Group
Sourced in United States
GGTI-298 is a laboratory reagent and research tool used in biochemical and cell biology studies. It functions as a geranylgeranyltransferase I (GGTase I) inhibitor. GGTase I is an enzyme involved in the post-translational modification of certain proteins. GGTI-298 can be utilized to investigate the role of GGTase I and protein geranylgeranylation in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Fti 277, by Merck Group (6 mentions)
Simvastatin, by Merck Group (3 mentions)
Farnesyl pyrophosphate, by Merck Group (3 mentions)
Geranylgeranyl pyrophosphate, by Merck Group (3 mentions)
Mevalonate, by Merck Group (3 mentions)
Atorvastatin, by Merck Group (2 mentions)
Cholesterol, by Merck Group (2 mentions)
Y 27632, by Bio-Techne (1 mentions)
Actinomycin d, by Merck Group (1 mentions)
Topotecan, by Merck Group (1 mentions)
Clodronate, by Merck Group (1 mentions)
C3 transferase, by Cytoskeleton (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Msto 211h, by American Type Culture Collection (1 mentions)
Nci h28, by American Type Culture Collection (1 mentions)
Ehmes 10, by American Type Culture Collection (1 mentions)
Fibronectin, by Thermo Fisher Scientific (1 mentions)
Vitronectin, by Merck Group (1 mentions)
Laminin, by Thermo Fisher Scientific (1 mentions)
F actin, by Thermo Fisher Scientific (1 mentions)
12 protocols using ggti 298
1
Modulating Rho GTPases in Human TM Cells
2
Comprehensive Mesothelioma Cell Line Protocol
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Human mesothelioma, MSTO-211H and NCI-H28 cells, were purchased from American Type Culture Collection (Manassas, VA, USA), and EHMES-1, EHMES-10 and JMN-1B cells were provided by Dr Hamada (Ehime University, Ehime, Japan). ZOL were purchased from Novartis (Basel, Switzerland), and FOH, GGOH, GGTI-298, ETO, topotecan and clodronate were from Sigma-Aldrich (St Louis, MO, USA). C3 transferase and NSC23766 were from Cytoskeleton (Denver, CO, USA) and Merck Millipore (Billerica, MA, USA), respectively. NE10790 were provided by Dr Ebetino FH (Warner Chilcott, Dundalk, Ireland). We purchased siRNA duplex targeting Cdc42 and non-specific control siRNA from Invitrogen (Carlsbad, CA, USA). Transfection of cells with the siRNA was conducted with Lipofectamine RNAiMAX (Invitrogen).
3
Antibody-Based Analysis of Cellular Signaling
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A list of the primary and secondary antibodies used in this study is presented in S2 Table . TRITC-phalloidin served for F-actin staining (Molecular Probes). DAPI and FITC-Dextran (FD405) were obtained from Sigma. Detection of active GTP-bound Ras and Rap1 in HUVECs was performed with Cell Signaling kits (#8821 and #8818, respectively). The low molecular Rap1 inhibitor GGTI298 was from Sigma. Control (SR-CL000-005) and Rasa3 (5’-GCGCTTTGGGATGAAGAAT-3’ and 5’CCTGAAGTTTGGAGATGAA-3’) siRNAs were purchased from Eurogentec. Tamoxifen was from Sigma, VEGF was from Peprotech, fibronectin and collagen I were from Gibco, vitronectin was from Millipore and laminin was from Invitrogen.
4
Receptor-Ligand Interactions in T Cell Activation
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Peptides (MCC, T102S, K3, and ER60), MHC, CD80, ICAM-1, ICAM-YFP, supported membranes, and T cells were prepared as described previously (12 (link), 36 (link), 41 (link)). CD80 fused to decahistidine-tagged SNAPf (GenScript) was expressed in ES-sf9 cells and purified by Ni2+-nitrilotriacetic acid agarose affinity (Qiagen) with 1 mM cysteine. T cells were transduced as described previously with Zap70-EGFP (12 (link)) or NFAT-GFP (42 (link)). All mouse work was approved by Lawrence Berkeley National Laboratory Animal Welfare and Research Committee under the Animal Use Protocol 17702. T cells were pretreated in suspension with Latrunculin A (Sigma) or GGTI-298 (Sigma) for 15 min or 1 h, respectively, before exposure to the lipid bilayer. Single-molecule and confocal imaging experiments were performed on separate motorized inverted microscopes as described previously (12 (link), 43 (link)). Single-molecule data were analyzed using a custom-written particle analysis suite developed in MATLAB (The MathWorks) and NFAT data were analyzed using ImageJ (44 (link)). More detailed methods are provided in SI Materials and Methods .
5
Small Molecule Inhibitor Procurement
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SAHA, FK228, CI994, CAY10481, PCI24781, PCI34051, and RGFP966 were purchased from Selleck Chemicals (Houston, TX, USA). GGTase-I specific inhibitor GGTI-298 was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-human GGTase-Iβ subunit and RhoA antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
6
Statin Drugs and Cholesterol Synthesis
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Nine statin drugs were obtained from commercial sources: Atorvastatin calcium salt trihydrate, simvastatin, pravastatin, mevastatin (PZ0001, S6196, P4498, M2537 Sigma, St Louis, MO, USA); fluvastatin, rosuvastatin, cerivastatin (F601250, R700500, C277000, Toronto Research Chemicals Inc., Toronto, ON, Canada); pitavastatin (S1759, Selleckchem, Boston, MA, USA); lovastatin (430-103-M050, Enzo life sciences, Farmingdale, NY, USA). We prepared 10 mM stock solution in DMSO for in vitro testing. For in vivo testing, pitavastatin was prepared as fresh suspension in methycellulose at 0.4 mg ml−1 (for oral gavage). fluvastatin, cerivastatin, and pitavastatin were prepared as a solution at 0.25 mg ml−1 in PBS for IP injection. GGTI-298 and FTI-277 (G5169, A1393, Sigma) were made into 10 mM stock solution in DMSO. AICAR (#S1802, Selleckchem) was made into 100 mM stock solution in PBS. Intermediate products of cholesterol synthesis were purchased from Sigma: mevalonolactone (M4667), farnesyl pyrophosphate ammonium salt (F6892), geranylgeranyl pyrophosphate ammonium salt (G6025), squalene (S3626), cholesterol solution (S5442), geranylpyrophosphate (G6772), and isopentenyl pyrophosphate triammonium salt solution (I0503).
7
Evaluation of FDA-Approved Drugs for Novel Activities
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Crizotinib, ceritinib, and lorlatinib were purchased from Selleck Chemical (Houston, TX). The library of FDA‐approved drugs (Screen‐Well FDA‐Approved Drug Library, 640 chemical compounds dissolved at 10 mM in dimethyl sulfoxide) was obtained from Enzo Life Sciences (Plymouth Meeting, PA). The following compounds were purchased from Sigma Chemical Co. (St. Louis, MO): cerivastatin, simvastatin, fluvastatin, atorvastatin, GGPP, FPP, squalene, GGTI‐298, FTI‐277, and verteporfin. Drug preparation and use of all reagents were conducted according to manufacturer's instructions.
8
Cellular Cholesterol Metabolism Assay
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Cell culture plastic ware, media, penicillin, streptomycin, and fetal bovine serum (FBS) were obtained from VWR (Toronto, ON, Canada). Secondary anti-rabbit and anti-mouse antibodies, propidium iodide (PI), simvastatin, mevalonate (Mev), farnesylpyrophosphate (FPP), geranylgeranylpyrophosphate (GGPP), GGTi-298, FTi-277, cholesterol, and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) (MTT), were obtained from Sigma (Sigma-Aldrich, Oakville, CA). Rabbit anti-human/mouse Rac1/2/3, RhoA, and Cdc42 were purchased from Cell Signaling (Canada). Casapase-Glo®-3/7, Caspase-Glo®-8 and Caspase-Glo®-9 assay were purchased from Promega (Toronto, ON, Canada). Tetramethylrhodamine, Methyl Ester, Perchlorate (TMRM) was purchased from Biotium (Hayward, CA, USA). Thin layer chromatographic plates (silica gel G, 0.25-mm thickness) were from Fisher (Winnipeg, Manitoba, Canada). Ecolite scintillant was from ICN Biochemicals (Montreal, Quebec, Canada). Unlabeled cholesterol standard was from Sigma-Aldrich (Oakville, ON, Canada). [1-14C]Acetate was from American Radiolabeled Chemicals (St. Louis, MO, USA). All other biochemicals were American Chemical Society grade and were obtained from either Sigma-Aldrich or Fisher Scientific (Winnipeg, MB, Canada).
9
Regulation of RhoGTPases in Osteoblasts
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Zoledronic acid was provided by Novartis (Basel, Switzerland), and atorvastatin, mevalonate, geranyl-geranyl-pyrophosphate (GGPP), farnesyl pyrophosphate (FPP), GGTI-298 and FTI-277 were obtained from Sigma-Aldrich (Munich, Germany). Rac1 inhibitors #1 (553502) and #2 (553511), Y-27632, rho kinase inhibitor (H-1152P) and Clostridium difficile toxin A were from Merck Chemicals (Darmstadt, Germany). Cdc42 inhibitor ML-141 was from Tocris Bioscience (Bristol, UK). Rho inhibitor #2 (BML-EI394) was obtained from Enzo (Lörrach, Germany). Rho inhibitor #1 (CT04) and Rho/Rac/Cdc42 activator I were from Cytoskeleton Inc. (Denver, CO, USA). Recombinant human DKK-1 was obtained from Peprotech (Hamburg, Germany). Antibody for RAP1A (sc-1482) was from Santa Cruz (Heidelberg, Germany), RAS (610001) antibody was from BD Biosciences (Heidelberg, Germany), the DKK-1 (MAB10962) antibody was from R&D Systems (Wiesbaden, Germany) and all other antibodies were obtained from Cell Signaling Technology (Frankfurt, Germany). DKK-1 small interfering RNA (siRNA; s22721 and s22723), Cdc42 siRNAs and nontarget siRNA were purchased from Applied Biosystems (Darmstadt, Germany). Cells were transfected using Dharmafect (Thermo Scientific, Waltham, Massachusetts (MA) USA).
10
Inhibiting γ-Secretase and Geranylgeranyl Transferase
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The γ-secretase inhibitor (GSI) (DAPT, Calbiochem, San Diego, CA), the geranylgeranyl transferase inhibitor (GGTI) (GGTI298, Sigma-Aldrich, Poole, UK), and the Rheo-switch ligand (RSL1, New England Biolabs Inc., Beverly, MA) were obtained commercially.
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