Protein Extraction and Quantification from Varicose Veins

Proteins were extracted from 31 varicose, and 34 control veins (100 µg) using RIPA buffer with protease inhibitor cocktails described earlier [6 (link)]. Protein concentrations were determined with the Bradford reagent as per manufactures instructions (Bio-Rad, Hercules, CA, USA). Total proteins were resolved on a 10% SDS-PAGE and transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). Membranes were blocked with 5% BSA in TBS for 1 h to reduce the non-specific binding of proteins, and membranes were incubated with primary antibodies (Supplementary Table S2) at 4 °C overnight. After rinsing thrice with TBS containing 0.1% Tween-20 (TBST), the membrane was incubated with HRP-conjugated secondary antibodies for 1 h at 37 °C in TBST. The chemiluminescence signal was detected using enhanced chemiluminescence (ECL) substrate (Bio-Rad, USA), and chemiluminescence measurements were performed using the Chemi Doc imaging system. Image J software was used for densitometry of immunoreactive bands.