circITGB6-pulled down proteins in A549 cells

A549 cells were transfected with 100 pmol DNA probes complementary to the back-splice site of circITGB6 or PDPN 3’UTR region. 24 h later, cells were washed with PBS and lysed with the lysis buffer (150 mM NaCl, 20 mM Tris-HCl, pH 7.5, 1.5 mM MgCl2, 0.5% NP-40, 0.5% Triton X-100, 10% glycerol, RNase inhibitors (Thermo Scientific), protease and phosphatase inhibitor cocktails). Pre-treated streptavidin magnetic beads were incubated with cell lysates at 4°C for 1 h, followed by washing five times with the washing buffer (20 mM Tri-HCl, pH7.5, 1 mM EDTA and 450 mM NaCl) and elution with Laemmli sample buffer. The circITGB6- or PDPN mRNA- interacting proteins were subjected to SDS-PAGE and visualized by coomassie blue staining or immunoblot analysis. Protein bands were excised and identified by LC-MS/MS mass spectrometry and Proteome Discoverer software (version 1.4; Thermo Scientific).