Bambi

Cells were split by radio immunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China) and supplemented with a 100 × protease inhibitor (Pierce, Rockford, IL, USA). The protein was separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, and then was transferred to a polyvinylidene Fluoride (PVDF) membrane (Millipore, Boston, MA, USA). Next, the protein was incubated with the corresponding primary antibody and secondary antibody. Antibodies included adipose triglyceride lipase (ATGL) (1:500, Cell Signaling, Boston, MA, USA cat. no. 2138), peroxisome proliferator-activated receptor γ (PPARγ) (cat. no. sc-7196), fatty acid binding proteins (aP2) (cat. no. sc-271529), fatty acid synthase (FAS) (cat. no. sc-21730), cyclin B (cat. no. sc-752), cyclin E (cat. no. sc-481), proliferating cell nuclear antigen (PCNA) (cat. no. sc-56), p21 (cat. no. sc-6246) (1:500 Santa Cruz, Dallas, TX, USA), cyclin D (1:500 BOSTER, China cat. no. BA0770), β-actin (1:500 Sungene Biotech, Tianjin, China), and BAMBI (1:500 Thermo Fisher Scientific, Waltham, MA, USA cat. no. PA5-38027).

Aortic valve (AV) specimens were collected at the time of surgery or from an explanted heart. Samples were immediately flash-frozen and stored at − 80 °C till used for RNA extraction. Total RNA from AV tissues was isolated using RNeasy Kit (Qiagen, Germantown, MD), according to the manufacturer’s instructions. The BAMBI gene expression level was validated by quantitative RT–PCR (qRT-PCR) using the Taqman method, as previously described^{90 (link)}. Briefly, cDNA was generated using 100 ng of total RNA and High Capacity cDNA Synthesize Kit (Catalog # 4,368,814 Thermo Fisher Scientific). Predesigned BAMBI (catalog # Hs03044164) and GAPDH (catalog # Hs02758991) primers were obtained from Thermo Fisher Scientific and the qRT-PCR were run in triplicates according to the manufacturer’s instructions. All Ct values were reported below 40, indicating acceptable PCR efficiency^{91 (link)}.

Briefly, 15 μg samples of total lysates from tissues or cells were run on a 10% SDS-PAGE gel and immunoblotted with the primary antibodies (1:1000) to BAMBI (ThermoFisher, PA5-38027), β-Tubulin (Sungene Biotech, KM9003), PPARγ (Abcam, ab3442), aP2 (Santa, Sc-271529), ATGL (CST, 2138), HSL (CST, 4107), Akt (CST, 4691), p-Akt (ser473,CST, 4060), UCP1 (Abcam, ab10983), PGC1α (CST, 2178), C/EBPβ (Santa, sc-7962), oxidative phosphorylation (OXPHOS) (Abcam, ab110413), and Nox4 (ABclonal, A11274). The intensity of bands was measured using ImageJ. All experiments were repeated at least three times and mean values were derived. All the uncropped blots are included in the Source Data file.