Tulp3

Briefly, VSMCs were washed by PBS twice followed by the addition of cell lysis buffer (ABGENT, Suzhou, Jiangsu, China). Then, collection of VSMCs lysates was performed and a BCA assay kit (MaiRuiBo, Chaoyang, Beijing, China) confirmed the concentrations of proteins. SDS-PAGE (10%) was for separating VSMCs lysates, and consequent proteins were transferred on PVDF membranes, followed by 5% BSA (in TBST buffer) blockade for 1 h. Afterwards, primary antibody against STAT3 (CST, Pudong, Shanghai, China), TULP3 (abcam) or GAPDH (Protein Tech Group, Wuhan, Hubei, China) was used for probing the membranes for 12 h at 4°C. Sequentially, matched secondary antibodies were employed for incubation. A Super ECL assay kit (YRBIO, Changsha, Hunan, China) was for protein band visualization.

Cells were lysed with RIPA buffer, and protein concentrations were determined using the BCA Protein Assay Reagent (Thermo Scientific, Waltham, MA, USA). Equal amounts of protein were subjected to 10% SDS-PAGE before being transferred to a nitrocellulose membrane. The membrane was incubated (overnight, 4 °C) with the following primary antibodies: (EN1-2377, NIBS, Beijing, China), (EN1-2378, NIBS, Beijing, China), (EN1 Polyclonal Antibody, Invitrogen, Waltham, MA, USA), (EN1-4D9, University of California, USA), (EN1-4G11, Columbia University, USA), (EN1, Abcam, Cambridge, MA, USA), (EN1, Sigma-Aldrich, USA), (EN1, Atlas Antibodies, Stockholm, Sweden), (EN1, Cusabio, Wuhan, China) (only EN1-2377 recognizes the correct size of EN1 band), (GAPDH, Origene, USA), and (TULP3, Abcam, Cambridge, MA, USA), (Gli1, Cell Signaling Technology, Danvers, MA, USA), before washing three times with TBST. The membrane was then incubated (1 h) with horseradish peroxidase-conjugated secondary goat anti-rabbit or anti-mouse antibodies (Cell Signaling Technology, Danvers, MA, USA), before being washed again with TBST (Table S3). Signals were detected using an ECL solution (Thermo Fisher, Waltham, MA, USA).