Antibodies that recognize phosphorylated or total proteins, including Src, p-Src (Y416), AKT, p-AKT (S473), S6, p-S6 (S240/244), ERK1/2, p-ERK1/2 (T202/Y204), FAK, p-FAK (T397), EGFR, p-EGFR (T1068), STAT3, STAT3 (T705) were purchased from Cell Signaling Technology (Beverly, MA). β-actin antibody and D-Luciferin bioluminescent substrate were purchased from Sigma-Aldrich. Saracatinib and capivasertib were purchased from Selleckchem (Houston, TX). Plasmid HA-AKT-DN (K179 M) (Addgene, Plasmid #16243) and HA-AKT-CA (Addgene, Plasmid #16244) were a gift from Dr. Mien-Chie Hung (Addgene, Plasmid #16243) [14 (link)]. Standard cell culture, plasmid transfection, Western blotting, colony formation, CellTiter-Glo® Luminescent Cell Viability and CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) assays were carried out as previously described [2 (link), 3 (link), 5 (link)].
Capivasertib
Manufactured by Selleck Chemicals
Sourced in United States
Capivasertib is a laboratory equipment product used for scientific research. It is a molecular compound with a specific chemical structure and properties. The core function of Capivasertib is to serve as a tool for investigating various biological processes and pharmaceutical applications, though its precise intended use is not provided.
Automatically generated - may contain errors
Lab products found in correlation
D luciferin bioluminescent substrate, by Merck Group (2 mentions)
Trametinib, by Selleck Chemicals (2 mentions)
Mk 2206, by Selleck Chemicals (2 mentions)
5 bromo 2 deoxyuridine brdu incorporation assay kit, by Roche (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Saracatinib, by Selleck Chemicals (1 mentions)
Ha akt dn k179 m, by Addgene (1 mentions)
β actin antibody, by Merck Group (1 mentions)
Niclosamide, by Selleck Chemicals (1 mentions)
Verapamil, by Bio-Techne (1 mentions)
Nicl2, by Bio-Techne (1 mentions)
Human tgf β1, by Thermo Fisher Scientific (1 mentions)
Sb431542, by Bio-Techne (1 mentions)
Dactolisib, by Selleck Chemicals (1 mentions)
Camptothecin, by Selleck Chemicals (1 mentions)
Ipatasertib, by MedChemExpress (1 mentions)
Alpelisib, by Targetmol (1 mentions)
Apitolisib, by Selleck Chemicals (1 mentions)
Torin1, by Bio-Techne (1 mentions)
Azd5363, by Selleck Chemicals (1 mentions)
13 protocols using capivasertib
1
Phosphoprotein Profiling in Cell Lines
2
Evaluating Small-Molecule Inhibitors on Cell Proliferation
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To gauge the effects of small-molecule inhibitors and calcium channel blockers on proliferation of the cells, a 5-bromo-2′-deoxyuridine (BrdU) incorporation assay kit (Roche Diagnostics GmbH, Mannheim, Germany) was performed as surrogate. In our study, MEK inhibitor trametinib, AKT inhibitors capivasertib and MK-2206, and STAT3 inhibitor niclosamide (all from Selleck Chemicals, Houston, TX, USA) were employed. DMSO was used as a solvent. Additionally, the effects of MgCl2, NiCl2, and verapamil (Tocris bioscience, Bristol, UK; all dissolved in DMEM/F12) were investigated. The cells (5 × 103/well) were seeded in 96-well half-area microplates at equal densities and allowed to adhere overnight in a complete culture medium. On the following day, the culture medium was replaced by a medium supplemented with inhibitors, MgCl2 or NiCl2 at the indicated doses. After an incubation period of 32 h, BrdU was added at a final concentration of 10 µM into the culture media. Another 16 h later, labeling was stopped, and BrdU uptake was measured according to the manufacturer’s instructions.
3
Quantifying Tumor Spheroid Growth and Necrosis
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Cells were seeded at 1000 cells/well in round‐bottomed ultra‐low attachment (ULA) 96‐well‐plates (VWR, #4441020) in 200 μL of the appropriate medium and grown in a CO2‐incubator at 37°C. Medium was changed every 2‐3 days. Images were acquired on the indicated days with an Olympus IX83 microscope, using a SP 10× objective and CellSens Dimension software. Spheroid area was determined in ImageJ software using freehand selection to mark spheroid outline, followed by area quantification in ImageJ. Necrotic core quantification (in three independent biological replicates) was performed using ImageJ software. Bright field images were analyzed using a macros function where images were converted to 8‐bit format, adjusted to Threshold (0.50) to define the necrotic core in each spheroid. Area in pixels2 was measured using “Analyze Particles” function with size of particles bigger than 2000 pixels2, and converted to mm2. In each experiment 4‐6 biological replicates were used. Where indicated, media were supplemented with SB 431542 (#1614, Tocris Bioscience), human TGFβ1 (#100‐21‐2UG, PeproTech), Capivasertib (AZD5363, #S8019, Selleckchem), or MK‐2206 (#S1078, Selleckchem).
4
Synthesis and Characterization of Compounds
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R1881 (synthetic androgen, also known as metribolone) and copanlisib were manufactured in‐house. Darolutamide was synthesized at Orion Corporation (Espoo, Finland). Apitolisib (S2696), dactolisib (S1009), capivasertib (S8019), and camptothecin (S1288) were from Selleckchem (Planegg, Germany). Alpelisib (T1921), duvelisib (T1988), and idelalisib (T1894) were from TargetMol (Wellesley Hills, MA, USA). Ipatasertib (HY‐15186) was from MedChemExpress (Monmouth Junction, NJ, USA).
5
Western Blotting Antibody Panel
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The following antibodies for the purpose of Western blotting were acquired from Cell Signaling Technologies: pAKT S473 (Catalog number 4060), PAN-AKT (#2920), Phospho-p44/42 MAPK (#4370), p44/42 MAPK (#4695), S6 Ribosomal Protein (#2317), Phospho-S6 Ribosomal Protein (#4858), Phospho-p70S6 Kinase Thr389 (#97596), p70S6 Kinase (#9202), Phospho-4EBP1 Thr37/46 (#2855), 4EBP1 (#9452). Antibodies against Beta-Actin were purchased from GeneTex (catalog number 109639). MAPK inhibitors were purchased from Tocris: FR180204 (catalog number 3706), U0126 (catalog number 1144), PD980595 (catalog number 1213). Sapanisertib (Cat# HY-13328) and Rapalink-1 (Cat# HY-111373) were purchased from MedChemExpress. Torin-1 (Cat #4247) was purchased from Tocris. PI3K/AKT/mTOR inhibitors BKM120 (Buparlisib; Novartis), AZD5363 (Capivasertib; Selleckchem), and RAD001 (Everolimus, MedChemExpress Cat# 159351–69–6) were generously provided by Dr Kathleen Millen.
6
Comprehensive Research Protocol Inventory
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AZ’1569 (compound-43), AZ’8037 (compound-25; ref. 11 (link)), AZD1480, and AZD6244 (Selumetinib/ARRY-142866) were obtained from AstraZeneca, 5-fluorouracil (5-FU) and oxaliplatin from the Belfast City Hospital Pharmacy, cetuximab from Merck Serono, and crizotinib from Pfizer. Ruloxitinib, capivasertib, ABT-737, and compound library were purchased from Selleckchem, S6K-18 and ABT-263 from Adooq Biosciences, and SN-38 from Abatra. See Supplementary Materials and Methods for references of non–FDA-approved drugs used. siRNA targeting BCL2L1 and the ON-TARGETplus siRNA library was obtained from Dharmacon. See Supplementary Materials and Methods for details of plasmids.
7
Evaluating Anti-Proliferative Effects of Afatinib and Capivasertib
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To gauge the effects of the EGF receptor antagonist afatinib and pan-AKT inhibitor capivasertib (both from Selleck Chemicals, Houston, TX, USA) on DNA synthesis, a 5-bromo-2′-deoxyuridine (BrdU) incorporation assay kit (Roche Diagnostics GmbH, Mannheim, Germany) was employed. For this purpose, the cells (HROG cells: 1 × 103/well; metastases cells: 3 × 103/well) were seeded in 96-well half area microplates at equal densities and allowed to adhere overnight in complete culture medium. On the following day, the culture medium was substituted with a medium supplemented with small molecule inhibitors (afatinib or capivasertib) at the indicated doses. After an incubation period of 32 h, BrdU labeling was initiated by adding labeling solution at a final concentration of 10 µM. Another 16 h later, labeling was stopped, and BrdU uptake was measured according to the manufacturer’s instructions using a GloMax-Multi Detection System (Promega, Madison, WI, USA).
8
Synthesis and Use of dTAG-47 Compound
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dTAG-47 was synthesized by the Vanderbilt Computational Structural and Chemical Biology Core Facility as previously described24 (link) and resuspended in DMSO. dTAG-47 was used at a final concentration of 500 nm for all experiments. Cycloheximide (Tocris) was resuspended in DMSO and used at 50 μg/mL for all experiments unless otherwise noted. Capivasertib (Selleckchem) was resuspended in DMSO and used at 500 nm for all experiments unless otherwise noted.
9
Antibody-based Protein Analysis Protocol
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Antibodies that recognize phosphorylated or total proteins, including AKT, p-AKT (S473), S6, p-S6 (240/244), and cleaved poly ADP-ribose polymerase (c-PARP), were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against β-Actin and CD31 were obtained from Abcam (Cambridge, MA, USA). 4′,6-diamidino-2-phenylindole (DAPI) and d -luciferin bioluminescent substrate were purchased from Sigma-Aldrich (St Louis, MO, USA). Capivasertib and LY294002 were purchased from Selleckchem (Houston, TX, USA).
10
Assessing Synergistic Drug Combinations
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Cells were incubated for 48 hours in the presence of increasing concentrations of single or combination drug treatments. Cell viability was assayed by adding Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc.) to the cell culture for the last 4 hours, and quantitated using Bio Tek Synergy H4 plate reader and Gen5 software as described previously (17 (link)). IC50 dose was defined as the IC50, calculated using nonlinear fitted dose–response curves in GraphPad Prism v.9.0 (RRID:SCR_002798). Experiments were done in triplicate. For in vitro experiments looking at the synergy between BH3 mimetics and TKIs, zero interaction potency (ZIP) synergy scores were calculated using SynergyFinder v.3.0 (https://doi.org/10.1093/nar/gkac382 ; RRID:SCR_019318). Venetoclax, navitoclax, A-1155463, dasatinib, ponatinib, BMS 345541, and capivasertib were obtained from Selleck Chemicals. NWP-0476 was provided by Newave Pharmaceutical, lnc.
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