Fig. 3

HNF4A-AS1 interacts with hnRNPU protein in NB cells. a Coomassie blue staining (left panel) and mass spectrometry (MS) assay (right panel) of indicated electrophoretic bands revealing the identification of protein pulled down by biotin-labeled HNF4A-AS1 in BE(2)-C cells. b Biotin-labeled RNA pull-down and Western blot assays showing the hnRNPU protein pulled down by sense or antisense (AS) HNF4A-AS1 from lysates of BE(2)-C cells. The HNF4A-AS1 AS- and bead-bound protein served as negative controls. c Dual RNA-FISH and immunofluorescence staining assay indicating the co-localization of hnRNPU and HNF4A-AS1 in the nuclei of SH-SY5Y cells stably transfected with empty vector (mock) or HNF4A-AS1. d RIP (upper right panel) and Western blot (lower right panel) assays using hnRNPU antibody showing the interaction between HNF4A-AS1 and hnRNPU protein in SH-SY5Y cells transfected with a series truncations of HNF4A-AS1 (left panel). The IgG-bound RNA was taken as a negative control. e Biotin-labeled RNA pull-down assay revealing the interaction between HNF4A-AS1 truncations and hnRNPU protein in BE(2)-C cells. Bead-bound protein served as a negative control. f RNA EMSA assay using biotin-labeled probes indicating the interaction of HNF4A-AS1 truncations with recombinant GST-tagged hnRNPU protein, with or without competition using an excess of unlabeled homologous probe. g Biotin-labeled RNA pull-down and Western blot assays showing the recovered hnRNPU truncations (upper panel) after incubation of biotin-labeled HNF4A-AS1 with full-length or truncated forms of GST-tagged recombinant hnRNPU protein (lower panel). h RIP assay using FLAG antibody indicating the interaction between HNF4A-AS1 truncations and FLAG-tagged hnRNPU protein in BE(2)-C cells. The IgG was applied as a negative control. Data are representative of three independent experiments in b–h