Hmga2

Human aortic smooth muscle cells after siRNA‐treated for 48 hours were harvested, and total protein was extracted using RIPA buffer (Solarbio) and 1 mM PMSF (Solarbio). Protein concentrations were determined using the Bradford method (Solarbio). Equal amounts of protein samples were separated by SDS‐PAGE and transferred onto a PVDF membrane (Merck) after electroblotting. The membrane was blocked with 5% non‐fat milk in PBS for 2 hours and then was incubated with the primary antibodies overnight at 4°C: HMGA2 (1:1000, Proteintech), PCNA (1:1,000, Wanleibio), SM22α (1:1,000, CUSABIO) and GAPDH (1:1,000, Wanleibio). After three 5‐minutes washes with TBS‐tween, the membrane was then incubated with goat anti‐rabbit IgG secondary antibody or rat antimouse secondary antibody (dilution at a 1:2000, Wanleibio) at 37°C for 2 hours. The membranes were visualized by ChemiDoc™ MP Imaging System (BIO‐RAD).

Cells were lysed with lysis buffer (50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 1% NP-40, 1 mM EDTA), protease inhibitor cocktail (Roche Applied Science), 1 mM PMSF, 1 mM NaF, and 1 mM sodium orthovanadate. Nuclear and cytoplasmic proteins were prepared as follows: cells were harvested and incubated in buffer A (10 mM Hepes, pH 7.8, 10 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 2 mg/ml aprotinin, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5% Triton X-100). After centrifugation, supernatants were collected as the cytoplasmic proteins. Buffer C (50 mM HEPES, pH 7.8, 420 mM KCl, 0.1 mM EDTA, 5 mM MgCl2, 10% glycerol, 1 mM dithiothreitol, 2 mg/ml aprotinin, and 0.5 mM phenylmethylsulfonyl fluoride) was added to the pellet. After rotation for 30 minutes and centrifugation, supernatants were collected as nuclear proteins. The following antibodies were used for immunoblotting and immunoprecipitation analyses: Flag-M2 (Sigma, F3165), Tubulin (T8328, Sigma), Histone H3 (sc-10809, Santa Cruz), T7 (A190-117A, Bethyl), HMGA2 (20795-I-AP, Proteintech), T7 Tag antibody agarose (69026, Novagen).

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).