Ecl chemidoc

Western blot analysis was performed on the proteins extracted from liver samples under each experimental condition. In brief, the liver tissues were physically homogenized and lysed in M-PER Mammalian Protein Extraction Reagent (Thermo Fisher, MA, 78,503) and centrifuged at 22,000 × g for 30 min at 4°C. The supernatant was used for the detection of HAMP or β-actin. The protein concentration was measured according to the Bradford method using bovine serum albumin (BSA) as a standard. The protein samples from all related experiments were processed in lithium dodecyl sulfate sample loading buffer (Bio-Rad, CA, 1,610,737), heated at 95°C for 5 min, loaded onto 10% Tris–glycine stain–free gel (Bio-Rad, 5,678,033), resolved by SDS-PAGE, and transferred to a polyvinylidene difluoride membrane using a Turbo Transfer System (Bio-Rad, 1,704,155).Specific antibodies directed to distinct HAMP (1:1,000; ThermoFisher, MA, PA5-90884) were used. The signals were detected by horseradish peroxidase (HRP)–based chemiluminescence (ThermoFisher, MA, 34,095), exposed to ECL Chemidoc (Bio-Rad, 1,708,280), and digitized using Image Lab software (Bio-Rad, 1,709,692).The methods have been described in detail in our earlier articles.