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. 2021 Jul 17;40(35):5403–5415. doi: 10.1038/s41388-021-01948-6

Fig. 6. UBE2CP3 mRNA was stabilized by IGFBP7 mRNA by forming a duplex RNA to interact with ILF3 protein.

Fig. 6

A The correlation (R value) of co-expression between each gene and UBE2CP3. B The IGFBP7 mRNA was highly co-expressed with UBE2CP3 mRNA. The P value was estimated using Pearson correlation test. C IGFBP7 expression was examined after silencing UBE2CP3 expression in GC cell lines by qRT-PCR assay. The P values were estimated using one-way ANOVA test. **P < 0.01. D The knockdown efficiency of IGFBP7 in GC cell lines was verified by qRT-PCR assay (left panel). UBE2CP3 expression was examined after silencing IGFBP7 expression in GC cell lines by qRT-PCR assay (right panel). The P values were estimated using one-way ANOVA test. **P < 0.01. E Schematic workflow of the RNA pull-down assay for identification of UBE2CP3 binding proteins. The sense (S) and anti-sense (AS) of UBE2CP3 RNA were biotinylated, refolded, and incubated with SGC7901 cell lysates. F Silver staining of the gel after SDS-PAGE of the potential UBE2CP3 binding proteins. Specific bands were excised and analyzed through mass spectrometry. The red arrow indicates gel band unique to UBE2CP3. The unique gel band was sent for mass spectrometry (MS) protein detection. G The top six protein hits according to the MS analysis of band labeled with a red arrow. H The sequence coverage of RNA binding proteins (ILF3) in the MS analysis was shown in the plot. I Single peptide-based protein identification of ILF3 protein. J Validation of UBE2CP3 binding proteins that obtained by RNA pulldown-MS method using western blotting. K Schematic workflow of the RNA Immunoprecipitation (RIP) assay for identification of UBE2CP3 binding proteins. L The transcripts abundances of UBE2CP3 and IGFBP7 were analyzed based on RIP-seq data. The ordinate represents counts per million (CPM) value (0–80). M The schematic diagram of the primer position of IGFBP7. N–P The transcript abundance of UBE2CP3, IGFBP7-5’UTR and IGFBP7-3’UTR was detected in the different group (IgG, anti-ILF3, input) of RIP assay by qRT-PCR. The P values were estimated using one-way ANOVA test. **P < 0.01.