Anti ddx17

Cellular extracts were prepared using lysis buffer containing 50 mM Tris HCL (pH 7.5), 250 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, and EDTA free protease inhibitor (Roche 11873580001). Extracts were run on a 4–12% Tris-Glycine gel (BioRad) and transferred onto PVDF membranes. Blots were blocked in 5% milk PBS-T for 1 h at room temperature followed by overnight incubation at 4 °C with primary antibodies at 1:1,000 (anti-HA Tag, Cell Signaling 3724S; anti-Flag Tag, Sigma F1804; anti-DDX5, Bethyl A300-523A; anti-DDX17, Bethyl A300-509A). Horseradish peroxidase (HRP) -conjugated secondary antibodies were used at 1:10,000 (anti-rabbit HRP, Santa Cruz sc-2030) or 1:1,000 (anti-mouse HRP, Cell Signaling 7076S).

After separation in SDS–PAGE (AA: 10%, AA/BisAA ratio: 36:1), the proteins were transferred onto PVDF membranes following overnight incubation with specific primary antibodies. The following primary antibodies were used: anti-actin (Sigma A5441, St. Louis, MO, USA), anti-E-cadherin (BD Biosciences, Heidelberg, Germany), anti-Zeb1 (Sigma AMAb90510), anti-CTBP2 (Abcam ab128871), anti-DDX17 (Sigma AV41029), anti-vimentin (Santa Cruz sc6260) and anti-N-cadherin (Cell Signaling 14215S). The secondary antibodies were from Sigma: anti-mouse (A9917) and anti-rabbit (A0545). Bound antibodies were visualized using the SuperSignal West Femto Maximum Sensitivity Substrate ECL kit (Thermo scientific, Boston, MA, USA), chemiluminescence was detected using ChemiDoc Touch Imaging System (Bio-Rad).