Horseradish peroxidase anti rabbit

For Western blot, 2 μg of purified complexes was run on NuPAGE 4 to 12% bis-tris gels (Thermo Fisher Scientific), transferred to Immobilon-P membranes (Millipore), and probed with anti-Strep (ab180957, Abcam, 1:5000) and anti-HA (3F10, Roche, 1:5000) antibodies in 5% milk–phosphate-buffered saline (PBS)/0.01% Tween overnight at 4°C. Membranes were then washed and incubated with horseradish peroxidase anti-rabbit (Santa Cruz Biotechnology, 1:40,000) and anti-rat (Jackson ImmunoResearch, 1:10,000) secondary antibodies, respectively, for 1 hour at room temperature. Immunoblots were developed using the Luminata Forte detection reagent (Millipore) and Hyperfilms ECL (GE Healthcare).

Western blotting was performed according to manufacturer’s instructions (Bio-Rad laboratories, Hercules, CA, USA). In brief, 20–40 μg of proteins was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Criterion TGX anykD acrylamide separating gel Bio-Rad). The separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore Corporation, Billerica, MA, USA) according to the manufacturer’s instructions. The membranes were dried and then incubated for 1h using primary antibodies for sirtuin proteins (SIRT1, SIRT2, SIRT3, SIRT4, SIRT6), Santa Cruz Biotechnology, Santa Cruz, CA, USA and glucokinase (GCK) Santa Cruz Biotechnology, Santa Cruz, CA, USA) in TBS (20 mM TRIS, pH = 7.6, 137 mM NaCl), 0.04% Tween-20 and 5% low fat milk and then incubated for 30 min with secondary antibodies conjugated with horseradish peroxidase (anti-rabbit, Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing with TBS, immunoblots were visualized by means of a chemiluminescence reaction (Millipore) by Image Lab^TM Software (Bio-Rad) under a luminescent image analyzer (Chemidoc XSR + Bio-Rad, Philadelphia, PA 19103, USA). Only bands below the saturation limit were analyzed and shown. β-actin (Sigma-Aldrich, S.r.l., Milan, Italy) was used as loading control.