Western blotting detection reagent

After HepG2 cells were treated with 1 μg/mL LPS in serum-free medium, they were exposed to various concentrations of DHP-3 (50-400 µM). Cells were scraped and collected by centrifugation, and cell pellets were lysed with the sample buffer containing 50 mM Tris-HCl (pH 6.8), 2% w/v sodium dodecyl sulfate (SDS), 10% glycerol, 5% 2-mercaptoethanol, and 0.1% w/v bromophenol blue. Cell lysates were sonicated in an ice bath for 15 sec, heated for 4 min at 100°C, separated by 10% SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane in a Tris/glycine transfer buffer. The membrane was blocked with PVDF Blocking Reagent (Toyobo, Tokyo, Japan), and then incubated with primary antibodies. After washing with phosphate buffered saline-Tween20 (PBS-Tween20), horseradish peroxidase-conjugated secondary antibodies goat anti-mouse IgG and goat anti-rabbit IgG were applied. The blots were developed using a western blotting detection reagent (Thermo Fisher Scientific, Waltham, MA, USA), and the band intensities were estimated using the VersaDoc™ Imaging System (Bio-Rad Laboratories, Hercules, CA, USA) and the iBright system (Thermo Fisher Scientific).