Ab126811

A total of 4 × 10⁵ cells were mixed with 1 ml RIPA solution (Sangon) to extract protein. Following denaturing in boiled water for 5 min, 10% SDS-PAGE gel was used to perform electrophoresis. PVDF membranes were used to perform gel transfer and blocking was performed for 2 h at room temperature in 5% non-fat milk. Blotting was performed using rabbit p-AKT (1:1200, ab18206, Abcam), AKT (1:1200, ab126811, Abcam), PI3K (1:1200, ab182651, Abcam), PI3K (1:1200, ab5451, Abcam), cytochrome c (1: 1200, ab90529, Abcam), and PI3K (1:1200, ab9485, Abcam) primary antibodies (overnight at 4°C) and goat IgG-HRP secondary antibody (1:800, MBS435036, MyBioSource). ECL (Sangon) was used to develop signal. Gray values were processed using Image J V1.6 software. Three independent replicates were set for each experiment.

Liver proteins were isolated with lysis buffer (pH 8.0, 50 mmol/L Tris-Base, 100 mmol/L NaCl, 5 mmol/L EGTA, 50 mmol/L Na₄P₂O₇, 1 M MgCl₂, 1% nonidet P/40, 0.3% Triton X-100, 0.5% sodium deoxycholate, and 1% protease inhibitor cocktail (catalog No. P8340, Sigma-Aldrich). Proteins were quantified by Bradfords method (13 (link)). A quantity of 50 μg of each protein sample was boiled and denatured in loading buffer containing 5% 2-mercaptoethanol (Invitrogen, USA). Samples were separated by electrophoresis on a 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were probed with phospho-Akt Ser473 (1:1,000; catalog No. ab109870; Abcam, USA). Pixel density was normalized to the total protein expression using the anti-Akt antibodies (1:1,000; catalog No. ab126811; Abcam). Proteins were detected using an enhanced chemiluminescence (ECL) substrate (Amersham Biosciences, UK), and protein expression levels are reported as a ratio of absorbance. For blots with labeling for more than one band (Akt), we performed measurements of the two bands together.

In all, 30 μg proteins from cell homogenates were loaded in a 3–8% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane, blocked for 1.5 h using 5% nonfat dry milk in TBS-t and incubated with rabbit anti-NRF2 primary antibody (1:1000; Abcam, ab137550), rabbit anti-phospho-Akt (Ser473) (1:1000; Abcam ab81283), rabbit anti-Akt (1:1000; Abcam, ab126811), rabbit anti-phospho-ERK1/2 (Thr202 + Tyr204) (1:250; Abcam, ab214362), rabbit anti-ERK1/2 (1:500; Abcam, ab196883), rabbit anti-β-actin (1:2000; Abcam, ab8227) overnight at 4 °C. Then, the membrane was incubated for 1.5 h with a goat HRP-conjugated anti-rabbit secondary antibody (1:2000; Abcam, ab205718). Bands were detected by the Clarity™ Western ECL Blotting Substrate using a ChemiDoc MP system (Bio-Rad Laboratories Inc) and quantified by the Image Lab™ Software.