Western Blot Analysis of Stemness Markers

Western blot was performed following the standard protocol [22 (link), 31 (link)]. The cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred to the polyvinylidene fluoride membranes. After the membranes were blocked, the diluted primary antibodies, MDR1 (MAB4163, 1:1000, Chemicon, Temecula, CA, USA ), ABCG2 (MAB4155, 1:1000, Millipore, Billerica, MA, USA), α-Tubulin (sc-5286, 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), E-cadherin (sc-7870, 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Snail (sc-28199, 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Nanog (sc-81961, 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Oct4 (GTX101497, 1:1000, Gene Tex, San Antonio, TX, USA), in Tris-buffered saline with Tween (TBST) buffer containing 3% non-fat milk were added to the membranes and incubated at 4°C overnight. On the second day, the goat anti-mouse conjugated secondary antibody was added at room temperature for 1 h. The immunoblots were developed using an enhanced chemiluminescence system and visualized on X-ray film.