Image labtmversion 6

Total proteins of hepatic tissues were extracted using a lysis buffer containing proteases, RIPA, and phosphatase inhibitors. Proteins were separated by electrophoresis and further electro-transferred onto a polyvinylidene fluoride membrane. Non-specific binding was blocked by adding 5% bovine serum albumin for 2 h at 24°C ± 2°C. Next, the membrane was incubated overnight at 4°C with anti-Akt (1:1000, Cell Signaling), anti-p-Akt (1:1000, Thermo Invitrogen, Shanghai, China), anti-PTRF (1:1000, Thermo Invitrogen, Shanghai, China), anti-PI3K) (1:1000, Abcam, Shanghai, China), and anti-GAPDH (1:1000, Cell Signaling, Boston, United States) primary antibodies in PBS + Tween buffer. The blots were then detected using an enhanced chemiluminescence kit (GE Biosciences, City of Saint Louis), United States), and the Bio-Rad Image LabTMVersion 6.0 software was used to analyze the density of the blots.

After SDS-PAGE electrophoresis, wet transfer was carried out to transfer the proteins from the gel to a polyvinylidene fluoride (PVDF) (Millipore, USA) membrane using Mini Trans-Blot^® Electrophoretic Transfer Cell (BioRad, USA). The primary antibodies used were NFκB p105/50 antibody, phospho-NFκB p105 (Ser933) antibody, PI3K p85 antibody, phospho-PI3K p85 (Tyr458)/p55 (Tyr199) antibody, and β-actin antibody (Cell Signaling, USA). All of the primary antibodies were diluted in 1:1000 except β-actin antibody which was diluted in a ratio of 1:1500. Chemiluminescence detection was done using ChemiDoc^TM XRS+ System (Bio-Rad, USA) and Immobilon Western Chemiluminescent HRP substrate (Millipore, USA). Image Lab^TM version 6.0 software (Bio-Rad, USA) was used for the quantification of the immunodetected protein bands. The expression of each protein was quantified and normalised against the protein expressions of their respective total protein and housekeeping gene, β-actin.