Following treatment for the indicated times, HAoSMCs were lysed with ice-cold IP lysis buffer supplemented with complete protease and phosphatase inhibitor cocktail (all from Thermo Fisher Scientific) [8 (link),35 (link),36 (link)]. Equal amounts of proteins were boiled in Roti-Load1 Buffer (Carl Roth) at 100°C for 10 min, separated on SDS/polyacrylamide gels and transferred to PVDF membranes. The membranes were incubated overnight at 4°C with primary antibodies: rabbit anti-SGK1 antibody (diluted 1:1000, #12103) or rabbit anti-GAPDH antibody (diluted 1:5000, #2118) and then with secondary anti-rabbit HRP-conjugated antibody (diluted 1:1000) (all from Cell Signaling) for 1 h at room temperature. For loading controls, the membranes were stripped in stripping buffer (Thermo Fisher Scientific) at room temperature for 10 min. Antibody binding was detected with ECL detection reagent (Thermo Fisher Scientific) and bands were quantified by using ImageJ software. Results are shown as the ratio of total protein to GAPDH, normalized to the control group.