Protein Isolation and Western Blot Analysis

Proteins were isolated from HAoSMCs using ice-cold Pierce IP lysis buffer containing complete protease and a phosphatase inhibitor cocktail (all from Fisher Scientific, Vienna, Austria). Protein concentration was measured using the Bradford assay (Bio-Rad Laboratories, Vienna, Austria) [37 (link),38 (link)]. Equal amounts of proteins incubated in Roti-Load1 Buffer (Carl Roth, Karlsruhe, Germany) for 10 min at 100 °C were separated on SDS-PAGE gels and transferred to PVDF membranes. Membranes were incubated with primary rabbit anti-RUNX2 (1:1000, Cell Signaling, Frankfurt am Main, Germany, #8486) or rabbit anti-GAPDH (1:1000, Cell Signaling, Frankfurt am Main, Germany, #2118) antibodies overnight at 4 °C and then with secondary anti-rabbit HRP-conjugated antibody (1:1000, Cell Signaling, Frankfurt am Main, Germany) for 1 h at room temperature, before being stripped in stripping buffer (Fisher Scientific, Vienna, Austria) at room temperature. Bands were detected with the ECL detection reagent (Fisher Scientific, Vienna, Austria) and quantified using the ImageJ software (NIH, Rockville, MD, USA, 1.52n). Data are shown as the ratio of total protein to GAPDH and were normalized to the control group [14 (link),21 (link)].