Fig 1: IGF2BP3 facilitates NOTCH3 mRNA stability in an m6A-dependent manner.A M6A RIP-qPCR assay analysis of the levels of m6A-modificated NOTCH3 in HONE1 and SUNE1 cells. B Endogenous RIP assays followed by qRT-PCR analysis examines the interaction between IGF2BP3 and NOTCH3 mRNA. C Schematic illustration of the putative wild-type m6A sites and designed mutant m6A sites in the NOTCH3 transcript. D RIP-qPCR assays showing the enrichment of NOTCH3 on IgG and IGF2BP3 in the NOTCH3-Wt or NOTCH3-Mut NPC cells. E Schematic illustration of mutated (GGAC to GGGC) sites in NOTCH3. F The luciferase activities of different mutated NOTCH3 reporters in the indicated groups. G Relative m6A level of NOTCH3 in NPC cells with or without METTL3 silencing or METTL3 inhibition. H RIP-qPCR assays show the enrichment of NOTCH3 mRNA on IgG and IGF2BP3 in NPC cells with or without METTL3 silencing or METTL3 inhibition. I The indicated SUNE1 cells were treated with actinomycin D (5 µg/ mL). RNA was isolated at the specified time point and NOTCH3 was subsequently analyzed by qRT-PCR analysis. The half-life of the mRNA was tracked by calculating its level relative to that in the untreated cell. J RIP assays followed by qRT-PCR to examine the interaction between CCR4 or CAF1 and NOTCH3 mRNAs in indicated NPC cells. K The luciferase activities of NOTCH3-Wt upon CCR4 or CAF1 overexpression compared to NOTCH3-Mut reporters or the addition of the METTL3 inhibitor in IGF2BP3-overexpressing NPC cells. Each error bar represents the mean ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ns no significance.
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