Anti ddx5

Protein samples were separated by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) followed by gel transfer to nitrocellulose membrane (Cytiva). The membranes were sequentially incubated with primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies followed by detection with enhanced chemiluminescence system (Millipore) using Amersham Imager 680 (Cytiva). The primary antibodies used in this study were anti-FLAG (Sigma-Aldrich, F3165, 1:8000 diluted), anti-GAPDH (ImB, MM001, 1:3000 diluted), anti-MeCP2 (Diagenode, pAb-052-050), anti-Rbfox1(Millipore, MABE159), anti-Rbfox2 (Bethyl, A300–864A), anti-Rbfox3 (Millipore, MAB377), anti-Matrin3 (Bethyl, A300–591A), anti-hnRNP M (Santa Cruz, sc-20001), anti-hnRNP A1 (Santa Cruz, sc-32301), anti-hnRNP C (Abclonal, A0057), anti-hnRNP H1 (Abclonal, A5924), anti-hnRNP U (Abclonal, A9907), anti-hnRNP F (Abclonal, A5505), anti-NF110 (Abclonal, A2496), anti-hnRNP K (Abclonal, A1701), anti-histone H3 (Abcam, ab1791), anti-DDX5 (Abcam, ab21696), and anti-GFP (Proteintech, 66002-1-Ig). Dilutions of all primary antibodies were 1:1000 if not specifically mentioned. HRP-conjugated secondary antibodies (1:2500 diluted) were anti-mouse IgG (Promega, W4021), anti-rabbit IgG (Promega, W4011), and conformation-specific anti-rabbit IgG (Cell Signaling, 3678).

Total proteins were extracted with ice-cold RIPA lysis buffer plus PMSF. Total protein concentrations were detected with BCA assay kit (Santa Cruz, California, USA). Prepared protein samples were separated in 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred into 0.22 μm PVDF membranes. After blocked with skim milk, these membranes were incubated with primary antibodies at 4 °C overnights, followed by incubation with the appropriate secondary antibody for 1 h at room temperature. Finally, the enhanced chemiluminescence (ECL, Pierce, Rockford, IL) visualized this membrane. The primary antibodies were anti-PCNA, anti-Ki-67, anti-Bcl-2, anti-Bax, anti-cleaved caspase-3, anti-cleaved caspase-9, anti-Cox-2, anti-MMP-2, anti-MMP-9, anti-DDX5 and anti-β-actin (Abcam, Cambridge, UK).