Enhanced chemiluminescence reagent

Total protein in tissues and cells was extracted using RIPA buffer (Beyotime, Shanghai, China) and determined using a BCA Protein Quantification Kit (Vazyme). Twenty micrograms of proteins were separated by 10% sodium dodecyl sulfonate-polyacrylamide gel (SDS-PAGE; Solarbio). Then the protein samples were transferred onto polyvinylidene difluoride membranes (Pall Corporation, New York, NYC, USA). Thereafter, the membranes were blocked with non-fat milk for 1 h and probed with primary antibody: hexokinase2 (HK2; ab209847; Abcam, Cambridge, MA, USA), pyruvate kinase isozyme type M2 (PKM2; ab137852; Abcam), FLOT2 (ab181981; Abcam), or GAPDH (ab9485; Abcam) overnight at 4 °C. After incubation with secondary antibody (ab205719; Abcam) for 2 h at room temperature, the protein bands were examined by an enhanced chemiluminescence reagent (Vazyme).

Aortic valve leaflets were collected and stored at −80 °C. For protein extraction, after removing large calcified nodules, leaflets were minced and homogenized in cold RIPA buffer containing PMSF using a tissue grinder (Tissuelyser, Jingxin, Shanghai). Protein concentration was determined with the BCA protein assay (cat #A55860, Thermo Fisher). For western blotting, equal quantities of proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. After blocking with skim milk, proteins were detected with specific primary antibodies followed by HRP-linked secondary antibodies. Finally, the blots were developed using the enhanced chemiluminescence reagent (cat #E411-04, Vazyme, Nanjing, China) with ImageQuant 800 (Amersham, GE healthcare); the bands were quantified by ImageQuant TL (Version 8.2, Amersham). The following primary antibodies were used at 1:1000 dilution otherwise specified: RUNX2 (cat #ET1612-47, Huabio, Hangzhou, China); ALP (cat #MAB1448, R&D); PAR2 (cat #ab180953, Abcam); PDK4 (cat #12949-1-AP, Proteintech); COL1A1 (cat #PA5-86949, Invitrogen); Fibronectin (cat #ab268020, Abcam); GPX4 (cat #ab125066, Abcam); GAPDH (cat #ET1702-66, Huabio, 1:5000); β-Actin (cat #ET1702-67, Huabio, 1:5000) and β-Tubulin (cat #ET1702-68, Huabio, 1:5000).

A total Protein Extraction Kit (Solarbio, Beijing) was used for protein extraction from wound tissues and macrophages, and the protein concentration was determined using a BCA assay kit (Solarbio, Beijing). After separation on a 10% SDS-PAGE gel, the proteins were blotted onto a polyvinylidene fluoride membrane (Bio-Rad Inc., USA). The membrane was blocked with TBST buffer containing 5% bovine serum albumin (BSA). After blocking, the membrane was incubated with the corresponding primary antibody at 4 °C overnight and incubated with the secondary antibody at room temperature for 1 h. Finally, all blots were investigated using an enhanced chemiluminescence reagent (Vazyme, China) and recorded using the Tanon luminescence imaging system.

Protein isolation was done using radioimmunoprecipitation assay buffer (CWBio, Beijing, China), and protein concentration was detected using a bicinchoninic acid protein assay kit (Tiangen, Beijing, China). Then, an equal amount of proteins was split by sodium dodecyl sulfonate-polyacrylamide gel (Solarbio) electrophoresis and blotted onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked using 5% nonfat milk for 1 h at indoor temperature. Next, the membranes were immunoblotted with primary antibodies against GAPDH (ab181602; Abcam), total p65 (t-p65; ab16502; Abcam), phosphorylated p65 (p-p65; ab86299; Abcam), B-cell lymphoma-2 (Bcl-2, ab196495; Abcam), BCL2-associated X (Bax, ab180733; Abcam), cleaved-caspase 3 (C-caspase 3, ab49822; Abcam), or total-caspase 3 (t-caspase 3, ab90437; Abcam) overnight at 4°C and then incubated with corresponding secondary antibody (ab6789; Abcam) for 1.5 h at indoor temperature. Finally, the protein bands were exposed through an enhanced chemiluminescence reagent (Vazyme) and analyzed by software Image J.