Odyssey infrared laser scan imaging instrument

RIPA buffer containing 10% phosphatase inhibitor (Beyotime, China) and 1% protease inhibitor (Beyotime, China) was used to lysate HRMECs and obtain proteins. The extracted protein concentration was measured using the BCA protein concentration assay kit (Beyotime, China). Protein samples of equal quality were resolved on 10% or 12% SDS-PAGE, then transferred to PVDF membranes, blocking the membrane with 5% skim milk at room temperature for 1 h, and the membranes were incubated at 4°C overnight with rabbit polyclonal antibody to SGLT-2 (Abcam, United States), BAX, Bcl-2, cleaved-caspase-3, ERK1/2, phosphorylated-ERK1/2, cPLA2 (Wanleibio, China), and rabbit polyclonal antibody to phosphorylated-cPLA2 (Cell Signaling Technology, United States). Mouse monoclonal antibodies to GAPDH (Abcam, United States) and β-actin (Abmart, China) were used as control. The membranes were incubated with the fluorescent secondary antibody (IRDye 800 CW goat anti-rabbit, 926-32211 or IRDye 680RD goat anti-mouse, 926-68070, 1:10000, LI-COR) at room temperature, dark for 1 h. The protein bands were scanned by using Odyssey Infrared Laser Scan-imaging Instrument (LI-COR). ImageJ was used for images analysis. All experiments were repeated three times.

Total protein of the rat liver was extracted by RIPA lysis buffer with 1% protease inhibitor and 10% phosphatase inhibitor. The concentration of protein was measured using the BCA kit and adjusted using RIPA lysis buffer. Then, the protein were separated through SDS-PAGE and transferred onto PVDF membranes. After blocking with 5% skimmed milk for 1 h, membranes were then incubated in antibody overnight at 4°C and incubated with the secondary antibody at room temperature for 1 h after being washed with PBST for 3 times. Then, the protein bands were detected with an Odyssey infrared laser scan-imaging instrument (LI-COR), and the images were analyzed using Odyssey Application Software 5.2.5.