Fig. 6

TRPM2-AS directly interacts with PABPC1. A FISH visualization revealing the subcellular localization of TRPM2-AS in HUVECs. Red fluorescence: Cy3-labeled TRPM2-AS; blue fluorescence: DAPI-stained nuclei. Scale bar: 20 μm. B RT-qPCR detecting the expression levels of TRPM2-AS in the cytoplasm and in nucleus respectively. C, D Schematic of RNA pull-down assay using biotin-labeled TRPM2-AS. The sense and antisense chains of biotin-labeled TRPM2-AS were synthesized as probes and combined with streptavidin-labeled magnetic beads. Proteins in HUVECs were extracted and incubated with beads coupled with RNA probe (C). RNA binding proteins were collected and stained with Coomassie bright blue. Mass spectrometry analysis confirming the proteins interacting with TRPM2-AS in HUVECs (D). E The expression level of NOTCH1 signaling pathway related proteins was identified by western blot with PABPC1/HSPA2/HSPA5/HSPA9/DDX41 knockdown. F The RNA secondary structure of TRPM2-AS was predicted using ViennaRNA Web Services (http://rna.tbi.univie.ac.at/) and RNA pull-down assays was conducted to confirm the domain of TRPM2-AS that interacted with PABPC1. G, H MYC-tag labeled full-length PABPC1 and its fragments I, II, III were overexpressed in HUVECs. RNA pull-down assay (G) and RIP assay (H) was used to confirm the exact fragment of PABPC1 that interacted with TRPM2-AS. Data were assessed with unpaired Student’s t test and presented as mean ± SD. ** P < 0.01