Anti gelsolin

Protein concentrations were determined by the BCA method. For Western blotting, total cell lysate was resolved by SDS-PAGE, transferred to PVDF membrane, and blocked with 5% non-fat dry milk in TBS-T. The membrane was incubated with primary antibody overnight at 4 °C, followed by peroxidase-conjugated secondary antibody for 2 h, and finally developed with the ECL system (Beyotime Institute of Biotechnology, Nanjing, China). Antibodies against phospho-eNOS (Ser1177) (Catalog No: 9570s), phospho-eNOS (Thr495) (Catalog No: 9574s), and eNOS (Catalog No: 32027s) were purchased from Cell Signaling Technology (Shanghai, China). Anti-GAPDH was from Tianjin Sungene Biotech Co., Ltd. (Tianjin, China) (Catalog No: KM9002T). Anti-Akt1/2/3 (Ser473) (Catalog No: sc-8312) and anti-p-Akt1/2/3 (Ser473) (Catalog No: sc-7985-R) were from Santa Cruz Biotech (Santa Cruz, CA). Anti-gelsolin (Catalog No: ab134183) was from Abcam (Shanghai, China). Western blotting results were quantified by densitometry and processed with the ImageJ software (National Institutes of Health software).

Aorta segment lysis was performed with chilled lysis buffer in presence of protease inhibitors. Lysate centrifugation was carried out at 12,000 rpm for 15 min at 4 °C. Total protein in the supernatant was quantitated with a BSA assay kit (P0006, Beyotime, Jiangsu, China). Protein separation was performed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and protein bands were electro-transferred onto polyvinylidene difluoride (PVDF) membranes. The samples were incubated overnight at 4 °C with anti-tropomyosin β, anti-transgelin, anti-annexin, anti-gelsolin, anti-HSP-27, anti-cofilin-1 and anti-GAPDH (1:1000; Abcam, Cambridge, UK) primary antibodies. Further incubation was performed with goat anti-mouse or anti-rabbit secondary antibodies (1:10,000; Santa Cruz Biotechnology, USA) for 1h in ambient conditions. Development was carried out with the BeyoECL kit (Beyotime, China) and a Tanon 5200 system.