Western Blot Analysis of Frozen Liver Tissue

Liver tissue stored at −80 °C was quickly placed in a mortar and liquid nitrogen was added, followed by pulverization of the material to obtain tissue powder. The powder was collected in a microcentrifuge tube for use in immunoblot analysis. The procedure for Western blotting was consistent with the method described by Tang et al. [59 (link)]. Experiments were performed using liver tissue samples with a protein concentration of 50 µg. Immunoreactive bands were colored with enhanced chemiluminescence (ECL, TransGen Biotech, Beijing, China), imaged using a FluorChem FC3 system (ProteinSimple, Waltham, MA, USA), and protein expression were quantified using Image Lab software. β-actin probed with antibody (Cincinnati, OH, USA) was used as a control.