Ecl plus

48 h after siRNA transfection, the cells were washed with PBS buffer and lysed with RIPA buffer (1× PBS, 1% IP-40, 0.5% sodium deoxycholate, and 0.1% SDS). After 1 h incubation on ice, the cellular mixture was centrifuged, and the supernatant was collected. Equivalent amounts (∼30 μg) of prepared cellular extracts were separated on a 10% SDS–polyacrylamide gel and transferred to a PVDF membrane (Bio-Rad). The membranes were probed with antibodies against human Polλ (Bethyl Lab) or Flag (Sigma-Aldrich), followed by appropriate secondary antibodies conjugated with horseradish peroxidase. The signals were detected using ECL-Plus (GenDEPOT). For the loading control, anti-β-tubulin antibody (Santa Cruz Biotechnology) or anti-lamin B1 antibody (Abcam) was used.

Total liver protein was extracted using RIPA lysis buffer (GenDEPOT, Katy, TX) supplemented with a protease inhibitor cocktail solution (GenDEPOT). The homogenate was incubated at 4°C for 2 h. The protein concentration was measured by the Bradford method. Protein samples (20 μg) were subjected to 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 2 h. They were then incubated at 4°C overnight with the primary anti-GAPDH antibody and anti-GLP-1R polyclonal antibody (1:1,000; Abcam, Cambridge, UK). After incubation with an anti-rabbit IgG horse radish peroxidase (HRP)-conjugated antibody (1:5,000; GenDEPOT) for 1 h, bands were detected using a chemiluminescent peroxidase substrate (ECL Plus; GenDEPOT) and imaged using the ChemiDoc XRS System (Bio-Rad, Hercules, CA).