Hmgb2

Cell lysis and protein extraction were carried out using the RIPA buffer (1 M Tris-HCl pH 8 (PanReac AppliChem, ITW Reagents), 0.5 M EDTA (Thermo Fisher Scientific), Triton X-100 (Sigma-Aldrich), 10% sodium deoxycholate (Sigma-Aldrich), 10% SDS (Sigma-Aldrich) and 3 M NaCl (Thermo Fisher Scientific)), supplemented with protease and phosphatase inhibitor cocktail (Sigma-Aldrich). Further, 20 µg of protein from each sample were separated by SDS-PAGE and transferred to a 0.2 µm pore-size nitrocellulose membrane (Bio-Rad). Membranes were blocked for 1 h with 5% BSA (PanReac AppliChem, ITW Reagents) in 1x TBS-T (0.1% Tween20, Bio-Rad) and incubated with specific antibodies HMGA2 (Proteintech, 20795-1-AP), HMGB1 (Abcam, ab18256), HMGB2 (Proteintech, 14597-1AP) and ɑ-tubulin (Sigma-Aldrich, T9026) overnight at 4 °C. Then, membranes were washed with 1x TBS-T and incubated with rabbit antimouse IgG (Sigma-Aldrich) or goat antirabbit IgG (Abcam, Cambridge, UK), both conjugated with peroxidase. HRP substrate (GE Healthcare, Life Sciences) was used for chemiluminescent detection, and image acquisition was performed using a Chemidoc Imaging System (Bio-Rad).