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GC376 Sodium

Manufactured by Aobious

GC376 Sodium is a laboratory equipment product. It is a chemical compound used in various analytical and research applications.

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3 protocols using GC376 Sodium

HEK 293T cells were co-transfected with either pmNG-Mpro-Nter-auto-NLuc or pmNG-Mpro-Nter-auto-L-NLuc plasmid along with either pLVX-EF1alpha-SARS-CoV-2-nsp5-2xStrep-IRES-Puro (Mpro WT) (Addgene plasmid # 141370) or pLVX-EF1alpha-SARS-CoV-2-nsp5-C145A-2xStrep-IRES-Puro (Mpro C145A) (Addgene plasmid # 141371) plasmid in 96-well white flat bottom plates at a ratio of 1:5 of sensor-to-protease plasmid DNA ratio. Transfected cells were concomitantly treated with a range of GC376 (GC376 Sodium; AOBIOUS—AOB36447; stock solution prepared in 50% DMSO at a concentration of 10 mM) concentrations. After 16 h of incubation with the inhibitor, BRET measurements were performed by the addition of furimazine (Promega, Wisconsin, USA) at a dilution of 1:200. The percentage activity was calculated by normalizing the BRET ratio with the negative control (No Mpro). Three independent experiments were performed in triplicates for each sensor construct.
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The human kidney epithelial cell line Lenti-X 293T was purchased from TaKaRa. The human liver cell line Huh7.5.1 was provided by Dr. Francis Chisari (Scripps Research Institute). The baby hamster kidney fibroblast cell line BHK21 (CCL-10), African green monkey kidney epithelial cells (Vero E6; CRL-1586), Caco-2 (HTB-37), Calu-3 (HTB-55), and A549 (CCL-185) were purchased from the American Type Culture Collection. A549-hACE2 (NR-53821) cells were obtained from BEI Resources. All cell lines were maintained in Dulbecco’s Modified Eagle Medium supplemented with 5% penicillin and streptomycin, and 10% fetal bovine serum (FBS) at 37°C with 5% CO2. The SARS-CoV-2 Nucleocapsid antibody (40143-MM05) was purchased from Sino Biological. The SARS-CoV-2 Nsp1 antibody (PA5-116941) was purchased from Thermo Fisher Scientific. The β-actin antibody (GTX109639) was purchased from Gentex. Secondary antibodies were purchased from LI-COR Bioscience. GC376 Sodium was purchased from Aobious (AOB36447). Remdesivir was purchased from MedChemExpress (HY-104077).
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The inhibitors at various concentrations (ranging from 10−3 to 10−10 M) were prepared from 10 mM stock solutions and incubated with 2 μM of recombinantly purified SARS-CoV-2 Mpro protease for 1 h at 37 °C in buffer containing tris-buffered saline (TBS), 1 M sodium citrate, 1 mM EDTA and 2 mM DTT, followed by the addition of cell lysates containing the BRET-based Mpro sensor [21 (link)]. GC376 (GC376 Sodium; AOBIOUS-AOB36447; stock solution prepared in 50% DMSO at a concentration of 10 mM) at a final concentration of 100 μM was used as a control. BRET measurements were performed at 37 °C by the addition of furimazine (Promega, Madison, WI, USA) at a dilution of 1:200. The bioluminescence (467 nm) and fluorescence (533 nm) readings were recorded using Tecan SPARK multimode microplate reader and used to calculate the BRET ratios (ratio of emission at 533 nm and 467 nm wavelengths). EC50 values were calculated using the BRET ratio obtained at 30 min after the addition of the NLuc substrate.
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