Pvdf membrane

Western blotting was used to measure the expression levels of Sirt1, liver kinase B1 (LKB1), AMP-activated protein kinase (AMPK) and p-AMPK proteins both in cell lines and in mouse liver tissues. In brief, cells or tissues were lysed and homogenized in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS, pH 7.4) supplemented with 1 × protease inhibitor cocktail, and the protein contents in the cell lysates were quantified by a BCA kit (Beyotime, China). Approximately 30 μg of protein sample was subjected to SDS-PAGE separation and was then transferred to a PVDF membrane (Millipore Corp, Bedford, MA). After blocking with 5% BSA in Tris-buffered saline with 0.1% Tween 20 (TBST), the PVDF membrane was rinsed and incubated with primary antibody (rabbit anti-Sirt1 antibody at 1:1000, rabbit anti-LKB1 antibody at 1:2000, rabbit anti-AMPK antibody at 1:1500, and rabbit anti-p-AMPK antibody and rabbit anti-GAPDH antibody at 1:1000, Cell Signaling Technology, USA) at 4 °C overnight. After vigorous washing with TBST 3 times, the PVDF membrane was then incubated with goat anti-rabbit IgG-HRP antibody (1:5000, Cell Signaling Technology, USA) for 1 h at room temperature. After washing with TBST, the membrane was then incubated with ECL substrate, and images were captured by a ChemiDoc MP imaging system (Bio-Rad, USA).

Western blotting and the analysis of the blot results was conducted as previously reported^{27 (link)}. In brief, cells were lysed and centrifuged at 12,000 g for 10 minutes at 4 °C. The supernatants were denatured at 100 °C for 5 minutes and mixed with a 1/5 volume (corresponding to the volume of the supernatants) of loading buffer (Beyotime, China). Then, 30-µg protein samples were loaded into each well in a 10% SDS-polyacrylamide gel and were subjected to electrophoresis. Once the proteins of diverse molecular weights were sufficiently separated, the proteins were transferred to PVDF membranes (Millipore, USA). Next, the PVDF membranes with the transferred proteins were blocked with 5% nonfat milk diluted in TBST for 2 hours at 25 °C and were then incubated with primary antibodies, such as SLC7A11, SLC3A2, GPX4, GSS, GCLC, ACSL4 (Cell Signaling Technology, USA) and β-actin (Beyotime, Shanghai, China), with dilutions of 1:1500, 1:2000, 1:2000, 1:2500, 1:3000 and 1:10,000, respectively, at 4 °C for 12 hours. Next, the membrane was washed again with TBST and was incubated with a 1:10,000 dilution of horseradish peroxidase (HRP)-labelled anti-rabbit IgG (Beyotime, Shanghai, China) for 1 hour at 25 °C. Finally, the respective protein bands were visualized using enhanced chemiluminescence reagents (BOSTER, Wuhan, China).