Anti ddx17

RNase-assisted RNA chromatography with RNAse A/T1 was performed, using in vitro-transcribed pri-mir-30c-1 and total MCF7 cell extracts28 (link). RNA-bound proteins were separated using 4–12% NUPAGE bis–tris system (Invitrogen) and individual lanes were subjected to mass spectroscopy (BSRC Mass Spectrometry and Proteomics Facility, University of St Andrews). Results were confirmed by western blot analysis with anti-SRSF3 (MBL), anti-PARP-1 (Santa Cruz), anti-DDX17 (Santa Cruz), anti-hnRNPA1 (Thermo Scientific) and anti-FUS (Abcam) specific antibodies, as indicated above (Western blot analysis section).

CRC cells were lysed in RIPA buffer (50 mM Tris-HCl pH 7.5, 1% NP-40, 0.25% sodium deoxycholate and 150 mM NaCl) supplemented with protease inhibitors (TargetMol, C0001). Lysates were loaded and separated by SDS–PAGE, transferred to a PVDF membrane (Millipore), and then incubated with the following antibodies: anti-DDX17 (Santa Cruz Biotechnology, sc-398168), anti-DDX5 (Cell Signaling Technology, CST, #9877), anti-CYBRD1 (Abcam, ab66048), anti-E-cadherin (CST, #3195), anti-Claudin-1 (CST, #13255), anti-Vimentin (CST, #5741), anti-N-cadherin (CST, #13116), and anti-β-actin (Sangon, D110001). On the next day, the membrane was probed with secondary antibodies at room temperature for 1 h, and protein bands were visualized using Immobilon™ Western Chemiluminescent HRP Substrate (Millipore). For RNA immunoprecipitation, pri-miR-149 and pri-miR-21 were in vitro transcribed and biotinylated by using T7 RNA polymerase and RNA 3’ End Desthiobiotinylation Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions, respectively. The biotinylated pri-miRNA was incubated with protein lysates from SW620 cells, immunoprecipitated with streptavidin beads and then subjected to western blot analysis by using anti-DDX17 antibody. Pri-miR-21 has been demonstrated to bind with DDX17 [24 (link)] and thus was used as a positive control.