Western lightning chemiluminescence reagent plus system

Whole-cell lysates of GSK-J4-treated cells were extracted with 300 μL of RIPA buffer (50 mM Tris, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate [SDS]), and subjected to western blot analysis. The membranes were incubated with polyclonal antibodies against UTX (ab36938, 1:2000, Abcam, Cambridge, MA, USA), Tri-methylation of histone H3 lysine 27 (H3K27me3) (A-4039, 1:2000, EpiGentek, Farmingdale, New York, USA), CDK4 (#12790, 1:1000, cell signaling, Danvers, Massachusetts, USA), Cyclin D1 (#2978, 1:1000, cell signaling, Danvers, Massachusetts, USA), E-cadherin (GTX124178, 1:5000, Genetex, Irvine, CA, USA), N-cadherin (sc-7939, 1:500, Santa Cruz Biotechnology, Santa Cruz, California, USA), Twist1 (#46702S, 1:500, cell signaling, Danvers, Massachusetts, USA), and β-actin (A5441, 1:10000, Sigma-Aldrich, St. Louis, Missouri, USA). Horseradish peroxidase-conjugated anti-rabbit secondary antibody was added to detect primary antibodies, and blots were developed with a chemiluminescence system (Pierce). All resolved protein bands were developed using the Western Lightning Chemiluminescence Reagent Plus system (Amersham Biosciences). All the experiments were repeated at least three times with similar results.

Whole-cell lysates of CD73-shRNA-treated cells were extracted with 300 μL of radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris, 150 mM sodium chloride (NaCl), 1% NP40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS)) and subjected to Western blotting analysis. The membranes were then incubated with polyclonal antibodies against CD73 (ab91086, 1:1000; Abcam, Cambridge, UK), E-cadherin (GTX124178, 1:5000; Genetex, Irvine, CA, USA), vimentin (ab92547, 1:1000; Abcam, Cambridge, UK), snail (#3879, 1:1000; Cell Signaling, Danvers, MA, USA), and β-actin (A5441, 1:10000; Sigma-Aldrich, St. Louis, Missouri, USA). Horseradish peroxidase-conjugated anti-rabbit secondary antibody was added to detect the primary antibodies, and the blots were developed using a chemiluminescence system (Pierce). All resolved protein bands were developed using the Western Lightning Chemiluminescence Reagent Plus system (Amersham Biosciences). All experiments were repeated at least three times, with similar results. The whole Western Blot figures can be found in the Supplementary Materials File.