Cells were plated at 2 × 105/well in 6-well plates. After 36 h, cells were infected with OVV-LG at an MOI of 0.001. After a 1-h incubation, E-MEM containing 0.8% methyl cellulose supplemented with 5% FBS and reagents was added to each well, and plaque sizes were calculated with a BZ-X700 microscope. Reagents used for plaque assays were as follows: nocodazole (Wako, Osaka, Japan), colchicine (Wako), cytochalasin D (Wako), Rhosin (Merck, Boston, MA, USA), ML-141 (Sigma, St. Louis, MO, USA), and NSC23766 (Abcam, Cambridge, UK).
Nsc23766
Manufactured by Abcam
Sourced in United Kingdom
NSC23766 is a small molecule inhibitor that targets Rac1, a Rho-family GTPase. It functions by blocking the interaction between Rac1 and its guanine nucleotide exchange factors, thereby inhibiting Rac1 activation.
Automatically generated - may contain errors
Lab products found in correlation
Rac1 pull down activation assay kit, by Cytoskeleton (2 mentions)
Ml141, by Merck Group (1 mentions)
Rhosin, by Merck Group (1 mentions)
Cytochalasin d, by Fujifilm (1 mentions)
Nocodazole, by Fujifilm (1 mentions)
Colchicine, by Fujifilm (1 mentions)
Z devd fmk, by R&D Systems (1 mentions)
Rhcxcl16, by R&D Systems (1 mentions)
2 meoe2, by Selleck Chemicals (1 mentions)
Sb203580, by Abcam (1 mentions)
Akti 1 2, by Abcam (1 mentions)
Ag1478, by Cell Signaling Technology (1 mentions)
Jwh133, by Bio-Techne (1 mentions)
Am630, by Bio-Techne (1 mentions)
Thp 1, by American Type Culture Collection (1 mentions)
Mcf 7, by American Type Culture Collection (1 mentions)
Cxcl12, by Thermo Fisher Scientific (1 mentions)
Jurkat, by American Type Culture Collection (1 mentions)
Catchpoint camp fluorescent assay kit, by Molecular Devices (1 mentions)
Ati2341, by Bio-Techne (1 mentions)
6 protocols using nsc23766
1
Plaque Assay for Oncolytic Virus
2
Optimizing Kidney Organoid Response
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To determine optimal concentration of each compound, PKHD1-mutant organoids were treated with the following concentration from day 16 to day 35 of differentiation: T-5224 (Thermo Fisher Scientific, 50-115-1835) at 5, 10, and 20 μM; Z-DEVD-FMK at 1, 10, and 50 μM (R&D Systems, FMK004); quercetagetin at 10, 40, and 200 μM (Millipore Sigma, 551590); NSC23766 at 25, 50, and 100 μM (Abcam, ab142161); rhCXCL16 at 5, 10, and 25 ng/ml (R&D Systems, 976-CX-025); 2-MeOE2 at 1, 5, and 10 μM (Selleckchem, S1233); R-naproxen at 10, 20, 200, and 400 μM (Millipore Sigma, 82170); and R-ketorolac at 0.5, 1, 10, and 20 μM (Millipore Sigma, 1654) (45 (link), 46 (link), 73 (link)–77 (link)). Full medium exchanges were conducted every 2 to 3 days. Treatment of the kidney organoids-on-a-chip was started at day 16 of their differentiation until day 35. The compounds were added to the medium reservoir at every medium change in the following concentrations: T-5224 at 10 μM, Z-DEVD-FMK at 1 μM, quercetagetin at 10 μM, NSC23766 at 10 μM, rhCXCL16 at 1 ng/ml, 2-MeOE2 at 5 μM, R-naproxen at 20 μM, S-naproxen at 20 μM, and R-ketorolac at 1 and 10 μM.
3
GPCR Signaling Modulation Assay
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The following drugs were used in this study: pertussis toxin (PTX; List Biological Laboratories Inc.); AG1478 (Cell Signaling Technology); ZCL278, EHT1864 (Cayman Chemicals); SP600125, Y27632 (Wako); NSC23766, SB203580, and Akti1/2 (Abcam). MCHR1:EGFP and SSTR3:EGFP clone cells were serum‐starved for 24 h prior to the pretreatment. Cells were pretreated with 120 ng/ml PTX for 24 h in serum‐starved medium. For other reagents, the final concentrations of the reagents and the pretreatment periods were: 10 µM AG1478 (30 min); 10 µM Y27632 (30 min); 50 µM ZCL278 (30 min); 10 µM EHT1864 (30 min); 3 µM NSC23766 (30 min); 30 µM SB203580 (30 min), 3 µM SP600125 (30 min), and 3 µM Akti1/2 (30 min).
4
Rac1-mediated Modulation of Tight Junctions
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Chemicals. JWH133 (cNR2 agonist), and AM630 (cNR2 antagonist) were purchased from Tocris Bioscience (Bristol, UK). JWH133 and AM630 were dissolved in dimethyl sulfoxide (DMSO) and then diluted with sterile PBS. NSC23766 (Rac1 inhibitor) was purchased from Abcam (cambridge, UK) and also dissolved in DMSO. Rac1 Pull-down Activation Assay kit was obtained from cytoskeleton Inc. (denver, cO, USA). Rat anti-Rac1 monoclonal antibody (cat. no. PA1-091; 1:1,000) was purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Anti-occludin (cat. no. sc-133256; 1:800), anti-ZO-1 (cat. no. sc-33725; 1:800) and anti-claudin-5 (cat. no. sc-374221; 1:800) antibodies were purchased from Santa cruz Biotechnology, Inc. (dallas, TX, USA). An antibody targeting β-actin (cat. no. 3700; 1:1,000) was purchased from cell Signaling Technology, Inc. (danvers, MA, USA). Goat anti-rabbit (cat. no. sc-2004; 1:2,000) and goat anti-mouse secondary antibodies (cat. no. sc-2005; 1:2,000) were purchased from Santa cruz Biotechnology, Inc. (dallas, TX, USA).
5
Phagocytosis Assay with Inhibitors
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ROCK inhibitor Y27632 (Calbiochem, La Jolla, CA, USA) is a selective inhibitor of the Rho-associated kinase p160 ROCK (an effector kinase of RhoA), NSC23766 (Abcam, Cambridge, UK) is an inhibitor of Rac1. Before starting phagocytosis assays with latex beads, as described above, MC were incubated with Y27632 at a concentration of 10µM for 15 minutes at 37°C, as described in [42] . NSC23766 was used at two different concentrations (10 and 50µM) for incubation of MC for 30 minutes at 37°C, as described in [43] .
6
Jurkat, THP-1, and MCF-7 Cell Culture
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The leukemic cell line Jurkat and the monocytic cell line THP-1 were purchased from the ATCC (Teddington, UK) and both cell lines were grown as described [5] . The breast cancer cell line MCF-7 was obtained from the ATCC and grown in DMEM containing 10% FCS and 2 mM L-glutamine. The chemokine CXCL12 was obtained from Peprotech (London, UK); CCL3 has been described previously [16, 27] .
NSC23766, AMD3100, and H89 were purchased from Abcam (Cambridge, UK).
ATI2341 was from Tocris Biosciences (Bristol, UK), EHT1864 was purchased from Cambridge Biosciences (Cambridge, UK). The anti-CXCR4 (12G5) antibody was from R&D Systems (Abingdon, UK) and the corresponding FITC labelled anti-mouse secondary antibody was from Sigma (Poole, UK). Rac1 Pull-down Activation Assay Kit was obtained from Cytoskeleton Inc (Denver, USA), the CatchPoint cAMP Fluorescent Assay Kit was from Molecular Devices (Wokingham, UK). All other chemicals were from Fisher Scientific (Loughborough, UK).
NSC23766, AMD3100, and H89 were purchased from Abcam (Cambridge, UK).
ATI2341 was from Tocris Biosciences (Bristol, UK), EHT1864 was purchased from Cambridge Biosciences (Cambridge, UK). The anti-CXCR4 (12G5) antibody was from R&D Systems (Abingdon, UK) and the corresponding FITC labelled anti-mouse secondary antibody was from Sigma (Poole, UK). Rac1 Pull-down Activation Assay Kit was obtained from Cytoskeleton Inc (Denver, USA), the CatchPoint cAMP Fluorescent Assay Kit was from Molecular Devices (Wokingham, UK). All other chemicals were from Fisher Scientific (Loughborough, UK).
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