Fuji film luminescent image analyzer las 4000

The protein extracts were separated on 10% SDS-PAGE gels and then transferred to polyvinylidene difluoride membranes (GE Healthcare, Buckinghamshire, UK). Western blotting49 (link) was performed using anti-GABAAR, anti-GAD1 and anti-GAD2 (Proteintech, Chicago, USA), anti-GABABR (Novus, Bangkok, Thailand), and anti-β-actin (Chemicon, Temecula, CA) antibodies. The membranes were subsequently incubated with an HRP-labeled goat anti-rabbit (or mouse) secondary antibody. The membranes were developed using an ECL Plus detection kit (GE Healthcare). The resulting bands were visualized and quantified using a Fuji Film Luminescent Image Analyzer (LAS-4000, Fuji Film Co., Tokyo, Japan) and analyzed using Multi Gauge Ver. 3.0 Image Analysis software (Fuji Film).

Mice lung tissue samples were homogenized in cold lysis buffer (50 mM Tris-HCl, pH 7.4, 5 mM EDTA, 0.5%NP-40, 150 mM NaCl) supplemented with 1x protease/phosphatase inhibitor cocktail (Cell Signaling Technology). Equal protein amounts of tissue lysates were loaded. Proteins were separated by SDS-PAGE gel and transferred to PVDF membranes. After blocking in 5% milk in TBST (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.1% (v/v) Tween-20) for 1 h, the membranes were incubated with iNOS, CTGF, α-SMA, and FN antibodies, respectively, overnight at 4°C, followed by the second antibody. The protein expression was determined using Lumigen ECL Ultra Western Blotting HRP Substrate (Lumigen), and the signals were detected by Fujifilm Luminescent Image Analyzer (LAS4000).