Anti zikv e

To investigate the antiviral effects of afatinib, erlotinib, sunitinib, and compounds 10b, L1, and L3, HEK-293 or MCF-7 cells were infected with DENV-1, -2, or ZIKV with or without (i.e., only solvent) the compounds. At the indicated time points after infection, the cell lysates were analyzed by Western blot with the antibodies anti-DENV NS3 (GeneTex, Irvine, CA, USA), anti-ZIKV E (GeneTex), anti-HER2 (Cell Signaling Technology, Danvers, MA, USA), anti-HER2 phospho Y877 (Cell signaling), anti-Src (GeneTex), anti-Src phospho Tyr418 (GeneTex), anti-ERK1/2 (Cell Signaling), anti-ERK1/2 phospho Thr/Tyr204 (Cell Signaling), anti-GAPDH (Millipore Corporation. Billerica, MA, USA), and anti-actin (Millipore). Then, the membranes were probed with horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibodies (Jackson ImmunoResearch, Suffolk, UK). The signals were developed by enhanced chemiluminescence (Millipore) and photographed using a Luminescent Image Analyzer (LAS-3000; Fujifilm Corporation, Tokyo, Japan).

Cells were harvested and lysed in sample buffer (50 mM Tris–HCl [pH 7.4), 1 mM PMSF, 10% glycerol, 6% SDS, 5% mercaptoethanol and 0.1% bromophenol blue), and the protein concentration was determined by BCA protein assay (Thermo Fisher Scientific, Rockford, IL). Briefly, protein bands separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were transferred to a PVDF membrane and then blocked with blocking buffer (5% non-fat milk in Tris-buffered saline). Specific protein bands were detected using the following primary antibodies: anti-ZIKV E (Gene Tex Inc, Alton Pkwy Irvine, CA), anti-ZIKV NS4A (Gene Tex Inc, Alton Pkwy Irvine, CA), anti-MAVS (Bethyl Laboratories, Montgomery, TX), anti-TRAF6, anti-HA, anti-Flag M2, anti-c-Myc, anti-β-actin (Sigma-Aldrich, St. Louis, MO), and anti-TBK1/NAK (Cell Signaling, Danvers, MA) antibodies. Further incubation with a horseradish peroxidase-conjugated secondary antibody and signals were detected by enhanced chemiluminescence using a commercial kit (Thermo Fisher Scientific, Rockford, IL) according to the manufacturer’s suggested protocol.