Total protein extracts were obtained by lysis of 5×105 cells in ice-cold lysis buffer supplemented with a cocktail of anti-proteases and anti-phosphatases as described.22 (link) Equivalent amounts of protein extracts were denatured, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Blots were incubated for 30 min in Tris-buffered saline containing 0.1% Tween 20% and 5% non-fat dry milk, and then overnight at 4°C with rabbit anti-NANOG (Santa Cruz, clone H155, cat# sc33759), anti-SNAIL1 (Cell Signaling, clone L70G2, cat# 3895S), anti-SOX2 (Cell Signaling, clone D6D9, cat# 3579), anti-GSK-3β (Cell Signaling, clone D75D3, cat# 5676) and antiphosphorylated (phospho)-GSK-3β ser9 (Cell Signaling, cat# 9336S) mAb, followed by appropriate secondary horseradish peroxidase-conjugated antibodies.
For quantification of total and active transforming growth factor (TGF)-β, tumor cells and CSC were plated at 1×107 cells in T75 culture flasks for 24 hours, then starved in 15 mL of serum-free medium for additional 48 hours. The conditioned medium was then harvested and concentrated 70× using Vivaspin Turbo 15 RC 10K (Startorius). Total and active TGF-β were measured using LEGENDplex multi-analyte assay kit according to the manufacturer’s instructions (Biolegend, cat# 740450).
For quantification of total and active transforming growth factor (TGF)-β, tumor cells and CSC were plated at 1×107 cells in T75 culture flasks for 24 hours, then starved in 15 mL of serum-free medium for additional 48 hours. The conditioned medium was then harvested and concentrated 70× using Vivaspin Turbo 15 RC 10K (Startorius). Total and active TGF-β were measured using LEGENDplex multi-analyte assay kit according to the manufacturer’s instructions (Biolegend, cat# 740450).