Fig. 5

CMTR1 interacts with splicing factor ELAVL1 in gastric cancer cells. a GO pathway analysis via Metascape program (https://metascape.org) of 204 CMTR1-interacting proteins derived from BioGRID database (https://thebiogrid.org). b Venn diagram showing the identification of CMTR1-binding partners via over-lapping analysis of CMTR1-interactng proteins derived from BioGRID database, established splicing factors, and RBPs binding with CD44 pre-mRNA in POSTAR3 (http://postar.ncrnalab.org) or ENCORI (https://rnasysu.com/encori/) database. c Co-IP and western blot assays indicating the interaction of CMTR1 with splicing factors in AGS cells. d Schematic depiction (upper panel) and cross-linking RIP assay (lower panel) showing the interaction of ELAVL1, HNRNPC, HNRNPU, or PTBP1 with CD44 pre-mRNA containing alternative splicing sites around exon 10 in AGS cells. e RT-PCR assay (left panel) and western blot (right panel) assays indicating the differential expression levels of CD44v6 and CD44s in gastric cancer AGS and MKN-45 cells stably transfected with empty vector (mock), ELAVL1, scramble shRNA (sh-Scb), sh-ELAVL1 #1, or sh-ELAVL1 #2. f Representative images (upper panel) and quantification (lower panel) of immunofluorescence assay showing the co-localization (arrowheads) of ELAVL1 with CMTR1 in HGC-27 cells. g BiFC assay indicating interaction between CMTR1 and ELAVL1 (arrowheads) within HGC-27 cells co-transfected with pBiFC-VN173-CMTR1 and pBiFC-VC155- ELAVL1, with nuclei staining by DAPI. h In vitro binding (left panel), co-IP (right panel) and western blot assays showing the interaction between GST-tagged CMTR1 and MBP-tagged ELAVL1 proteins, and that in AGS cells transfected with full-length or truncations of HA-tagged CMTR1 and Flag-tagged ELAVL1 constructs. Fisher's exact test for over-lapping analysis in b. Data are representative of three independent experiments in c-h