CD44v6 Knockdown and 5FU+Silibinin Effects

Protein was extracted from CD44v6 knockdown and 5FU+ Silibinin treated CD44+ HCT116 cells and the concentration was determined by BCA protein estimation kit (Sigma-Aldrich, USA). 50 μg of protein was resolved on 12% SDS-PAGE and transferred to PVDF membrane by wet electro-blotting method at 4 °C. The membrane was blocked with 5% non-fat milk in Tris buffered saline for 3 h at room temperature followed by overnight incubation with primary the primary antibodies: anti-CD44v6 (1:250), anti-PARP (1:1000), LC-3 (1:1000) and anti-β-actin (1:1000) at 4 °C. Membrane was probed with horseradish peroxidase (HRP) conjugated secondary antibodies (1:10,000). Proteins were detected using EZ-ECL kit (Clarity Western ECL substrate, BIO-RAD, USA) according to manufacturer’s instructions and signal was captured on Kodak X-Omat blue film (NEN Life Sciences, Inc., Boston, MA) in the dark^{54 (link)}.