Enhanced chemiluminescent solution

Proteins were extracted from tumor tissues collected from control (untreated) and treated groups by using RIPA lysis buffer (50mM Tris-HCL, pH 8.0, with 150 mM sodium chloride, 1.0% Igepal CA-630 (NP-40), 0.5% sodium deoxychlorate, and 0.1% sodium dodecyl sulfate) with protease inhibitor and 500mM phenyl methyl sulfonyl fluoride (51 (link)). Protein estimation was carried out by using BCA Protein Assay Reagent Kit (PIERCE, Rockford, IL). Equal amounts of supernatant protein (50 μg) from the control and treatment groups were denatured by boiling for 5 min in SDS sample buffer, and were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes for immunoblotting. Membranes were blocked with 5% skim milk in Tris-buffered saline buffer containing Tween 20 [10mM Tris-HCL (pH 7.6), 150 Mm NaCl, and 0.5% Tween 20] and were probed with Sox2, CD44s {CD44 (156-3C11) Mouse mAb #3570}, which recognizes the epitope centered around Pro210 of human CD44 (UniProt ID P16070), MMP9, Survivin, Caspase 3, Cyclin D1, p-STAT 3, p-STAT 3, ABCG2 and ABCC1 antibodies (Cell Signaling and Technology) in 1:1000 dilutions. Horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) were used. Proteins were visualized by using enhanced chemiluminescent solution (Biorad) and were exposed to Chemidoc Instrument (Biorad).