Enhanced chemi luminescence (ecl)

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.

Total protein was extracted from liver tissue, using RIPA protein lysis buffer containing 1 mM PMSF. 300 ng total protein were separated by 10% SDS-PAGE gel at 80 V for 1 h, transfered with polyvinylidenedifluoride (PVDF) membrane at 100 V for 1.5 h, blocked in 5% BSA (Solarbio, Beijing, China) and probed with appropriate primary antibodies against the target proteins. PTEN (Cat:#9188S), PI3 Kinase p85 (Cat:#4292S) anti bodies were purchased from Cell Signaling (Beverly, MA, USA). p-Akt (Cat:66444-1-Ig) and β-actin (Cat:20536-1-AP) anti bodies were purchased from Proteintech (Protein tech, Chicago, IL, USA). These were followed by incubation with Goat Anti-Rabbit immunoglobulin (Ig) G (H+L) (Cat:SA00001-2) (Protein tech, Chicago, IL, USA), and antigen-antibody complexes were visualized using the chemilucent ECL (TransGen Biotech, Beijing, China) detection system. The densitometric analysis conducted by Image J software 1.6.0 (National Institutes of Health, Bethesda, MD, USA) [37 (link),38 (link),39 (link)].