β tubulin

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).

Cellular proteins were extracted from TNBC cells using RIPA lysis buffer containing 1 mM PMSF and a cocktail. Following extraction, the proteins underwent electrophoresis and were subsequently transferred to PVDF membranes. Then TBST-dissolved 5% skim milk powder was blocked for 2 h at room temperature; after blocking, the membranes were exposed to the relevant antibodies and incubated overnight at 4 °C, FTSJ1 (1:1000, abcam (Waltham, MA, USA), ab227259), β-tubulin (1:1500, Huabio (Beijing, China), M1305-2), GAPDH (1:1000, Cell Signaling Technology (Danvers, MA, USA), #5174). These membranes were then incubated with the corresponding secondary antibodies for 2 h at room temperature. An appropriate amount of ECL luminescence working solution (Adansta, San Jose, CA, USA) was uniformly added. Image Lab 6.0.1 software (Bio-Rad, Hercules, CA, USA) was used for scanning development, and the strip scan map was saved. The grayscale values of the blots were quantified using ImageJ 1.50i (USA).