Fig. 4

LINC00571 interacts with HNRNPK protein and ILF2 protein. A Silver staining unveiled proteins interacting with LINC00571, with biotin-labeled sense or antisense LINC00571 probes used for RNA-protein pull-down against MDA-MB-231 cell lysates. B A simplified flowchart outlined the systematic screening process used to identify proteins that interacted with LINC00571. C Mass spectrometry analysis revealed HNRNPK peptides and ILF2 peptides pulled down by LINC00571 sense probes. D Immunoblot analyses were performed for HNRNPK and ILF2 on biotin-labeled sense and antisense LINC00571 probe pull-down eluates from MDA-MB-231 and BT-549 cell lysates, with GAPDH as a loading control. E RNA immunoprecipitation (RIP) was conducted on MDA-MB-231 and BT-549 cells using HNRNPK and IgG antibody or ILF2 and IgG antibody. The precipitates underwent immunoblot analysis with HNRNPK and GAPDH antibody or ILF2 and GAPDH antibody. HNRNPK or ILF2 enrichment of LINC00571 relative to IgG enrichment values was quantified by qRT-PCR. F RNA-FISH and immunofluorescence staining assays revealed subcellular co-localization of LINC00571 (green), ILF2 (red), and HNRNPK (cyan), along with nuclear staining using DAPI (blue). scale bar: 10μm (G) Schematic representation of HNRNPK with functional protein domains. HNRNPK had been truncated within regions: 1-143aa, 1-213aa, 144-213aa, 214-463aa, and 144-463aa. H-I Relative enrichment of endogenous LINC00571 in truncated HNRNPK RIP was measured by qRT-PCR, following MDA-MB-231 cells transfected with 3xFlag-HNRNPK truncations. Statistical analyses are depicted in bar graphs. Data are presented as mean ± SD from three independent experiments. Significance levels are denoted as * for p<0.05, ** for p<0.01, and *** for p<0.001, as determined by the t-test