Fig. 4

MNX1-AS1 directly binds to PKM2 and importin α5. a RNA protein pull-down assays using biotin-labelled sense or anti-sense MNX1-AS1 probes against cell lysates from HepG2 cells. PKM2 and importin α5 were subsequently identified by using mass spectrometry. b Biotin-labeled sense (S) or antisense (AS) MNX1-AS1 probes were used for RNA-protein pull-down against HepG2 (left) and PLC/PRF/5 (right) cell lysates. c and d RIP assays conducted in HepG2 cells against PKM2 (C) or importin α5 (D) with samples analyzed by Western blotting and semi-quantitative RT-PCR against MNX1-AS1. e and f RNA pull-down assays were conducted with biotin-labelled sense or anti-sense MNX1-AS1 adsorbed to streptavidin-conjugated beads against the indicated GST-tagged-PKM2 or His-tagged-importin α5 proteins purified from E.coli. Results were assessed by Western blotting. g Electrophoretic Mobility-Shift Assays (EMSA) analysis of the interactions between biotin-labelled MNX1-AS1 (2 nM) and recombinant GST-tagged-PKM2 or His-tagged-importin α5 proteins. Signals were revealed by Streptavidin-HRP. Unlabeled MNX1-AS1 (10 μM) was used as competitor. h HepG2 cells co-transfected with FLAG-importin α5 and HA-PKM2 were used to perform sequential immunoprecipitations with anti-FLAG and anti-HA antibodies. i Structure plot of MNX1-AS1 based on minimum free energy predicted by Vienna RNA. Exon1 (E1, nt 1–503, blue) and exon 2 (E2, nt 504–1041, orange) are color highlighted. j Subcellular fractions of HepG2 cells were analyzed by Western blotting to determine the subcellular localization of PKM2 and importin α5. k Fluorescence in situ hybridization of MNX1-AS1 followed by immunofluorescence staining of PKM2 and importin α5 in HepG2 cells. Cell nuclei were decorated by DAPI (A-H, J and K) are represent of three independent experiments