Oseltamivir carboxylate

Neuraminidase (NA) inhibition assay was performed using the NA-Fluor influenza Neuraminidase Assay Kit (Life Technologies, Carlsbad, CA, United States) performed according to the manufacturer’s instructions. EBL was serially diluted from 1000 μg/ml to 1 μg/ml in 96-well black plates. As a positive control, oseltamivir carboxylate (AOBIOUS INC, MA, United States), a specific NA inhibitor, was used from 10 to 0.01 μM. Influenza A/PR/8/34 virus was added to each well and incubated for 30 min at 37°C. The mixtures were incubated with 200 µM NA-Fluor Substrate for 1 h at 37°C and terminated by NA-Fluor stop solution. The reaction was monitored using a fluorescence spectrometer (Promega, Madison, WI, United States), with an excitation wavelength of 365 nm and an emission wavelength of 445 nm.

A neuraminidase (NA) inhibition assay was performed using the NA-Fluor influenza Neuraminidase Assay Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. IQC was serially 2-fold diluted from 100 μM to 1.56 μM in 96 well black plates. As a positive control, oseltamivir carboxylate (Aobious Inc, MA, USA), a specific NA inhibitor, was used from 10 μM to 0.0001 μM. H1N1 or H3N2 (2 × 10⁶ PFU) IAV was added to each well and incubated for 30 min at 37 °C. The mixtures were incubated with 200 µM NA-Fluor Substrate for 1 h for 37 °C and terminated using NA-Fluor stop solution. The reaction was monitored by a fluorescence spectrometer (Promega, Madison, WI, USA) with an excitation wavelength of 365 nm and an emission wavelength of 445 nm.

Oseltamivir carboxylate was purchased from AOBIOUS Inc. (Gloucester, MA, USA). Antibodies targeting influenza proteins, PA, NA, NP, PB1, PB2, M1, and NS-1, were procured from GeneTex (San Antonio, TX, USA). Anti-β-actin was purchased from Cell Signaling Technology (Cell Signaling Technology, Boston, MA, USA).