Fig. 5

SLCO4A1-AS1 binds to TOX4. A Overview of the in vitro RNA pull-down assay and identification of lncRNA SLCO4A1-AS1 associated cellular proteins. B Silver staining of biotinylated SLCO4A1-AS1 associated proteins. The SLCO4A1-AS1-specific bands (arrows) were excised and analyzed by mass spectrometry (MS), which were identified as TOX4. C Western blotting for proteins from SLCO4A1-AS1 pull-down assays. D Western blotting of TOX4 in samples pulled down by full-length (fragment A, including Exon1-4) or truncated SLCO4A1-AS1 (fragment B–D). Exon mapping showed that SLCO4A1-AS1 exon 3 and exon 4 interacted with TOX4 and was indispensable. E The PC9 cell lysates were immunoprecipitated with control rabbit IgG or anti-TOX4 antibody, and complexes were analyzed for enrichment of SLCO4A1-AS1 by RT-qPCR. The data are presented as mean ± SD (***p < 0.001). Specific immunoprecipitation of TOX4 was confirmed by western blotting (Inset). F TOX4 enriched SLCO4A1-AS1 was confirmed in SLCO4A1-AS1-expressing H1299 cell lysates. RT-qPCR data are presented as mean ± SD (***p < 0.001; ns, not statistically significant). Specific immunoprecipitation of TOX4 was confirmed by western blotting (Inset). G Western blot quantification of cytosolic and nuclear protein levels of TOX4 in H1299-TOX4 and CL1-0-TOX4 cells. H Immunofluorescence staining showed that TOX4 was localized in the nucleus. Scale bar = 20 μm. I RNA fluorescence in situ hybridization (RNA-FISH) and immunofluorescence staining showed that SLCO4A1-AS1 co-localized with TOX4 in SLCO4A1-AS1-expressing H1299-TOX4 cell line. Scale bar = 20 μm