Briefly, 30 μg protein from each sample was resolved by SDS-PAGE on Tris-Glycine gels, and transferred to PVDF membrane. Membranes were blocked with 5% (v/v) bovine serum albumin in Tris-buffered saline containing 0.1% (v/v) Tween 20 (TBST) and incubated with primary antibodies overnight at 4°C. Membranes were washed three times for 5 min with TBST, incubated in either HRPgoat-anti-mouse (ab6789, Abcam) or HRP-goat-anti-rabbit (ab6721, Abcam) secondary antibodies for 1 h at room temperature. Immunoreactive bands were visualized using Pierce ECL plus Western blotting substrate (32132, Thermo Fisher Scientific) .
For nuclear Nrf2 accumulation assays, HUVECs and HAOECs were harvested and lysed to obtain cytoplasmic and nuclear lysates using the Keygen Nuclear-Cytosol Protein Extraction Kit from Nanjing KeyGen Biotech. Co., Ltd. (Nanjing, China).
The primary antibodies used to probe the membranes included Nrf2 (1:1000), Fyn (1:2000) and GAPDH (1:5000) were purchased from Santa Cruz Biotechnology, Santa Cruz; Akt and p-Akt (Ser473; 1:2000), GSK3B and p-GSK3B (Ser9; 1:2000), c-Caspase 3 (1:1000) were purchased from Cell Signaling Technology; 3-nitrotyrosine (1:2000), Bax (1:2000) and Bcl-2 (1:2000) were purchased from Abcam. The expression of specific antigens was quantified using ImageQuant 5.2 software (Molecular Dynamics), and the expression of GAPDH was used as loading control.
For nuclear Nrf2 accumulation assays, HUVECs and HAOECs were harvested and lysed to obtain cytoplasmic and nuclear lysates using the Keygen Nuclear-Cytosol Protein Extraction Kit from Nanjing KeyGen Biotech. Co., Ltd. (Nanjing, China).
The primary antibodies used to probe the membranes included Nrf2 (1:1000), Fyn (1:2000) and GAPDH (1:5000) were purchased from Santa Cruz Biotechnology, Santa Cruz; Akt and p-Akt (Ser473; 1:2000), GSK3B and p-GSK3B (Ser9; 1:2000), c-Caspase 3 (1:1000) were purchased from Cell Signaling Technology; 3-nitrotyrosine (1:2000), Bax (1:2000) and Bcl-2 (1:2000) were purchased from Abcam. The expression of specific antigens was quantified using ImageQuant 5.2 software (Molecular Dynamics), and the expression of GAPDH was used as loading control.