*2.6. Western Blot Assay*

Confluent MDCK cell monolayers were first infected with PR8 (MOI = 1), and then treated with brevilin A or vehicle. At the indicated time of 4 or 8 hour-post-infection (hpi), proteins from total cell lysates were separated using SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Millipore, Burlington, MA, USA). The membrane was blocked with 5% nonfat milk or 5% BSA in PBS containing 0.1% Tween 20 and incubated overnight at 4 ◦C with primary mouse anti-HA (1:1000), mouse anti-NA (1:1000), mouse anti-M1 (1:3000), mouse anti-M2 (1:1000), rabbit anti-NS1 (1:1000), rabbit anti-NS2 (1:1000), and mouse anti-NP (1:1000) antibody, or mouse anti-GAPDH and mouse anti-β-actin antibody as control. The membrane was washed five times for 5 min with PBS-Tween and incubated for 1 h at room temperature with the respective secondary antibodies conjugated to horseradish peroxidase (HRP). After five washes for 5 min with PBS-Tween buffer, the chemiluminescence of the labeled proteins was visualized with HRP substrate and captured using the LICOR Odyssey imaging system. The relative densities of proteins were all determined by using ImageJ (NIH) v.1.46r (Wayen Rasband, US National Institutes of Health, Bethesda, MD, USA).
