Horseradish peroxidase conjugated anti rabbit igg or anti mouse igg

Total proteins were extracted from SNU-638 cells and HPSECs using radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific) supplemented with a protease inhibitor cocktail (GenDEPOT). Cells were sonicated for 2 min and centrifuged at 14,000× g for 10 min at 4 °C to remove insoluble cell debris. Protein concentration was determined using the BCA protein assay kit (Pierce, Rockford, IL, USA). Total protein lysates (30 μg) were separated via SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA, USA). After being blocked with 5% BSA, the membranes were incubated with primary antibodies against Sirt1 (sc-74504, 1:500, Santa Cruz Biotechnology, Dallas, TX, USA), α-tubulin (sc-5286, 1:500, Santa Cruz Biotechnology), lamin A/C (sc-376248, 1:500, Santa Cruz Biotechnology), MDM2 (#86394S, 1:1000, Cell Signaling Technology, Inc., Beverly, MA, USA), and Sirt6 (ab191385, 1:1000, Abcam). The blots were then incubated with horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG (Thermo Fisher Scientific). Signals were visualized using the Clarity Western blot ECL Substrate (Bio-Rad, Hercules, CA, USA) and imaged using the ChemiDoc Touch Imaging System (Bio-Rad).

Immunoblot analysis was carried out as a previously described method [48 (link)]. Samples from right cerebral cortexes were homogenized in lysis buffer [1 % Triton X-100 and 1 mM EDTA in PBS (pH 7.4)] using a tissue homogenizer and centrifuged at 16,000 g for 20 min at 4°C. Supernatant was isolated and protein concentration of each sample was calculated with bicinchroninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s manual. Protein (30 µg) from each sample was loaded on 10 % sodium dodecyl sulfate poly-acrylamide gel electrophoresis and electrophoresed until dye went down to the bottom. Protein was transferred to a poly-vinylidene fluoride membrane and membranes were incubated with 5 % skim milk in tris-buffer saline containing 0.1 % Tween-20 (TBST) for 1 h. Membranes were washed three times with TBST for 10 min and reacted with following primary antibodies: anti-parvalbumin (1:1,000; Thermo Fisher Scientific) and anti-β-actin (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. They were washed with TBST and incubated with horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG (1:5,000; Thermo Fisher Scientific) for 2 h. Membranes were washed with TBST and reacted with enhanced chemiluminescence Western detection reagents (GE Healthcare) according to the manufacturer’s manual.