Horse radish peroxidase conjugated goat anti rabbit igg secondary antibodies

EPCs were solubilized using a lysis buffer (Beyotime Institute of Biotechnology), supplemented with 0.5 mM phenylmethylsulfonyl fluoride and centrifuged at 12,000 x g for 15 min. Protein concentrations were assayed using a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology), 30 µg total protein from each group was separated by sodium dodecyl sulfate-polyacrylamide gel (10% for nAChR and 5% for SIRT1) electrophoresis and these proteins were transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% bovine serum albumin, and probed with antibodies against sirtuin 1 (SIRT1; dilution, 1:200; cat. no. sc-15404; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), alpha-7 nicotinic receptor (nAChR-α7; dilution, 1:500; cat. no. AB15332; Merck Millipore, Darmstadt, Germany) and β-actin (dilution, 1:1,000; cat. no. AA128; Beyotime Institute of Biotechnology) at 4°C overnight. Following this, horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibodies (dilution, 1:1,000; Beyotime Institute of Biotechnology) were incubated at 37°C for 1 h, and bands were detected using an enhanced chemiluminescence kit (cat. no. 32109; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Densiometry signals were quantified by ImageQuant TL software (GE Healthcare Life Sciences, Chalfont, UK).