Horseradish peroxidase hrp conjugated anti mouse or anti rabbit immunoglobulin g

A total of 15 µg per well of proteins were boiled with 1 × loading buffer (Thermo Fisher Scientific, Waltham, MA, USA, cat. no. 39000) for 10 min. Proteins were loaded onto 10% sodium dodecyl sulfate–polyacrylamide gels and separated by electrophoresis, then transferred to polyvinylidene difluoride membranes. After that membranes were blocked with 5% bovine serum albumin (BSA) for 1 h and incubated overnight at 4 °C with primary antibodies against β‐actin (1:20,000; Santa Cruz Biotechnology, Inc), SGLT1 (1:1000; Alomone labs), SGLT2 (1:1000; Alomone labs), p53 (1:1000; Santa Cruz Biotechnology, Inc), p21 (1:1000; Santa Cruz Biotechnology, Inc), Nox2 (1:1000; Proteintech), p22^phox (1:1000; Santa Cruz Biotechnology, Inc), eNOS (1:1000; BD Biosciences) diluted in 5% BSA. After washing, membranes were incubated with relevant secondary antibody [horseradish peroxidase (HRP)‐conjugated anti-mouse or anti-rabbit immunoglobulin G (1: 20,000; Cell Signaling Technology)]. Finally, the membranes were washed and treated with enhanced chemiluminescence substrate (GE Healthcare, Little Chalfont, UK, cat. no. RPN2232) and visualized with a chemiluminescence system (UVItec, Cambridge, UK). For densitometric analysis of each blot, Image J software was used, and expression levels of each protein were determined in three independent experiments.