Sh 9000

Eph4 cells were cultured on Transwell permeable supports for 14 d as described above. To examine the transepithelial flux of fluorescent tracers, the cell layer on the Transwell permeable support was placed into an Ussing chamber with a 5 µm-diameter pore. The basal side of the chamber was filled with 3 ml of Solution A (140 mM NaCl, 5 mM glucose, 5 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂ and 10 mM HEPES-NaOH [pH 7.4]). The apical side of the chamber was filled with 3 ml of Solution A containing the fluorescent tracer fluorescein (Sigma-Aldrich) or FD-4 (final concentration, 0.5 mM) (Sigma-Aldrich). The temperature was maintained at 37°C and 100% O₂ was bubbled through the solution continuously. We collected 200 μl of the solution in the basal side of the chamber every 20 min from 0 to 80 min. The fluorescence of the collected sample was determined at an excitation wavelength of 495 nm by a microplate reader SH-9000 (Hitachi) and then the transepithelial flux for the fluorescent tracer was calculated.

The HEK 293T cells were seeded one day prior to transfection on a 3.5 cm glass bottom or normal culture dish with 5 × 10⁵ cells/2 mL. A total of 0.7 μg each of the pcDNA-PA, -PB1, and -PB2 (or PB2 mutant) plasmids was co-transfected with 0.6 μg of pcDNA-NP and 0.3 μg of pPolI-mScarlet plasmids using Lipofectamine LTX (Thermo Fisher Science, Waltham, MA, USA) and Opti-MEM (Thermo Fisher Science) according to the manufacturer’s instructions. At 48 h post-transfection (hpt), red fluorescence images were taken using a fluorescence microscope (Eclipse Ti2, Nikon, Tokyo, Japan), and cells were lysed in 100 μL of lysis buffer (50 mM Tris-HCl, pH 8.0, 200 mM NaCl, 0.5% Nonidet P-40 [Sigma-Aldrich, St. Louis, MI, USA], 1 mM DTT, 1 mM PMSF, 25% glycerol, and 1× protease inhibitor cocktail [Fujifilm]) at 4 °C for 60 min. The lysates were analyzed using a fluorescence microplate reader (SH-9000, Hitachi, Tokyo, Japan).