Fig. 4

LINC00460 interacts with IGF2BP2 and DHX9 protein. a LINC00460 intracellular localization was visualized in CRC cells: HCT116, SW480, by FISH analysis. b, c Relative LINC00460 levels in HCT116 and SW480 cell cytoplasm or nucleus were detected by qRT-PCR. U6 was used as nuclear control and β-actin was used as cytoplasm control. d Coomassie Blue staining of biotinylated LINC00460-associated proteins is shown (1: DHX9; 2: IGF2BP2, The proteins was identified by mass spectrometry). e, f The interactions between LINC00460,DHX9 and IGF2BP2 identified by RNA-Pull down assays and Western blots in HCT116 and SW480 cells. Biotin labeled LINC00460 and its antisense RNA were in vitro transcribed by using T7 RNA polymerase. GAPDH was used as a negative control. g, h, i, j Results from RIP and subsequent qRT-PCR assays. RIP assays were performed to detect the interactions of LINC00460 and IFG2BP2/DHX9 in HCT116 and SW480 cells. Relative quantification of LINC00460, GAPDH, and 18S rRNA in RNA-protein complexes immunoprecipitated with IgG or DHX9, IGF2BP2 antibodies from whole cell extracts. Relative expressions were normalized by the input RNA, GAPDH and 18 s rRNA were used as negative control binding RNAs. Error bars indicate mean ± SD, n = 3 for technical replicates. ***p < 0.001