Anx a5

Cells were washed with phosphate buffered saline (PBS) and whole cell protein lysates were collected in RIPA buffer (15 mM Tris-HCl, pH 7.6, 1% Igepal CA-630, 0.5% sodium deoxycholate, and 0.1% SDS), supplemented with a protease inhibitor cocktail (Pierce-ThermoFisher). Samples were separated on 10% Bis-Tris gels (Invitrogen), transferred onto 0.2 µM nitrocellulose membranes, and blocked in 5% non-fat milk in Tris-buffered saline. Proteins were detected with antibodies directed against AnxA2 (1∶1000, Santa Cruz), Anx A5 (1∶1000, Santa Cruz), and α-tubulin (1∶1000, Cell Signaling). A secondary goat anti-rabbit antibody linked to horseradish peroxidase (1∶1000, Jackson ImmunoResearch) was used before developing in ECL (Invitrogen).

Total protein concentration was calculated by the BCA Protein Assay kit (Pierce, Rockford, IL, USA), according to the manufacturer's instructions. The cells and tissues were lysed in cell lysis buffer for 20 min vibration on the ice and centrifuged at 12.000 × g at 4°C for 15 min. Proteins were separated upon 10% SDS-PAGE and transferred onto 0.45 mm PVDF membrane (Bio-Rad, California, Hercules, USA). The membranes were incubated with 5% nonfat milk powder which dissolved in TBST (20 mM Tris-HCL, pH 7.5, 150 mM NaCl, 0.1% Tween 20) for 2 hours. The following protein antibody Nrf2 (68.0 kDa), Histone H3 (15.0 kDa), and GAPDH (40.2 kDa) (Cell Signaling Technology, CST); HO-1 (34.6 kDa), NQO-1 (30.0 kDa), and GST (24.0 kDa) (Abcam); and ANXA5 (36.0 kDa) (Santa Cruz, CA) were used to bind with corresponding proteins and incubated overnight at 4°C. All these protein antibodies were diluted by the Antibody Dilution Buffer (Beyotime, China) with a ratio of 1 : 1000-2000. HRP-conjugated secondary antibody (1 : 4000, Cell Signaling Technology, USA) was used to bind with primary antibodies for about 1.5 hours. Finally, immunoreactive bands were visualized with electrochemiluminescence reagent (Amersham, Uppsala, Sweden). Densitometry and quantitative analysis were analyzed using the ImageJ software (Bethesda, USA) and regulated by Histone H3 and GAPDH.