Subcellular localization of ELDR and ELDR-interacting proteins. (A) Detection of ELDR in the THP-1 cell line ELDR-C1 with smFISH revealing its predominantly nuclear localization; ELDR (white spots), DAPI staining (blue). Scale bar, 10 μm. (B) smFISH analysis with IMARIS image software for 3D reconstructions. ELDR is located predominantly in the nucleus (red spots), but also outside the nucleus (yellow spots). DAPI staining (blue). Scale bar, 10 μm. (C) Cell fractionation followed by RT-qPCR for ELDR confirms its predominant nuclear localization. Results are presented as percentage of RNA level. GAPDH was used as a positive control for the cytoplasmic RNA fraction, and MALAT1 was used as a positive control for the nuclear RNA fraction. n = 3 biological replicates. (D) Cell fractionation followed by RT-qPCR for ELDR reveals its percentage in the indicated cell compartments. (E) Proteins detected by ChIRP-MS with biotinylated oligos against ELDR in ELDR-C1 and ELDR-C3 cell lines ordered according to fold enrichment than the EV control cell line. (F) GO, KEGG, and REACTOME pathway analyses of ELDR-associated proteins found in the CHIRP-MS experiment. (G) Extracts of the ELDR-overexpressing cell line ELDR-C1 were incubated with biotinylated oligos covering ELDR. RNA-protein complexes were precipitated with streptavidin beads and analyzed by Western blot with antibodies against the indicated proteins. Actin and HDAC2 are controls that do not bind to ELDR. (H) RNA immunoprecipitation with antibodies against the indicated proteins from ELDR-overexpressing cells analyzed by RT-qPCR with primers covering ELDR or the U1 RNA as a control. Data are presented as mean + SD from 3 independent experiments. ∗∗∗P < .001; ∗∗∗∗P < .0001. P values were calculated using parametric unpaired t test (panels C, H). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GO, gene ontology.