Anti ap1

Total protein was extracted from HAECs and vascular tissues derived from rats. Nuclear extracts were isolated from HAECs using a nuclear protein extraction kit (Beyotime, Shanghai, China). Protein samples were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% skim milk, the membranes were incubated with the appropriate primary antibodies including anti-TXNIP (1:1000; Proteintech), anti-TRX (1:2000; Proteintech), anti-AP1 (1:1000; Proteintech), anti-REF1 (1:1000; Bimake), anti-GAPDH (1:5000; Proteintech), anti-PCNA (1:2000; Proteintech), anti-NOX4 (1:1000; Proteintech), anti-NOX2 (1:1000; Santa Cruz Biotechnology), anti-SOD2 (1:2000; Santa Cruz Biotechnology), anti-eNOS (1:1000; Cell Signaling Technology), anti-p-eNOS (ser 1177) (1:1000; Cell Signaling Technology), anti-AKT (1:1000; Cell Signaling Technology), and anti-p-AKT (ser 473) (1:500; Santa Cruz Biotechnology) overnight at 4 °C. Then, the membranes were incubated with species-matched secondary antibodies (1:5000; Proteintech) for 1.5 h at room temperature. The blots were developed with a hypersensitive ECL chemiluminescence kit (Beyotime) and examined using a Bio-Rad gel imaging system (Bio-Rad, CA, USA).