USP31 Antibody - #DF9989

Figure 5CircARCN1 affects the interaction between HuR and USP31 mRNA, and down-regulates USP31. (A) Venn diagram analysis of all mRNAs negatively correlated with circARCN1 in our RNA-Seq data (n = 9, data in Figure 1A and B), HuR target mRNAs identified by THP-1 FLASH-Seq, HuR target mRNAs in the ENCORI database (ClusterNum > 100), and HuR consensus target genes from a previous study.36 USP31 was identified to interact with HuR and was negatively correlated with circARCN1 in RNA-Seq data. (B) Correlation analysis of circARCN1 with USP31 (RNA-Seq data in Figure 1A and B, n = 9). (C) Correlation analysis of circARCN1 with USP31 in PBMC samples (n = 45), by RT-qPCR. (D) RT-qPCR analysis of USP31 mRNA levels after circARCN1 knockdown or overexpression in THP-1 macrophages. The results demonstrated that circARCN1 could regulate USP31 mRNA levels. (E) Western blot analysis of USP31 protein levels after circARCN1 knockdown or overexpression in THP-1 macrophages. The results demonstrated that circARCN1 could regulate USP31 protein levels. (F) FLASH-Seq, using the anti-HuR antibody, performed in THP-1 macrophages, with circARCN1 overexpression or knockdown, identified two BSs (BS1 and BS2) of HuR in the 3′UTR of USP31 mRNA. (G) Diagram showing USP31 3′UTR lacking BS1 (△1), BS2 (△2), or both BSs (△1–3). RNA pull-down assays were performed in THP-1 lysate, followed by western blot analysis, comparing the binding capacity with HuR among these mutants. (H) RIP experiment was conducted in THP-1, using anti-HuR antibody, followed by RT-qPCR analysis, showing the interaction between HuR and USP31 mRNA. (I) RIP assays comparing USP31 mRNA-binding capacity among HuR deletion mutants (HuR△1, HuR△2, and HuR△3). (J) RIP assays using anti-HuR antibody were conducted in THP-1, with circARCN1 knockdown or overexpression. CircARCN1 knockdown enhanced the interaction between HuR and USP31 mRNA, while circARCN1 overexpression weakened their interaction. (K and L) RNA stability assay. RNA synthesis was blocked with 5 μg/mL actinomycin D for indicated hours in THP-1. HuR knockdown in THP-1 resulted in decreased USP31 mRNA stability (K). CircARCN1 knockdown increased USP31 mRNA stability, while HuR silencing could abolish this effect (L). (M and N) Western blot analysis was conducted to examine the impact of USP31 silencing on the effects of circARCN1 knockdown in THP-1 macrophages (M). Protein levels were quantified in three independent experiments, as depicted in the bar-diagrams (N). The results demonstrated that USP31 silencing counteracted the effects of circARCN1 knockdown on NF-κB quiescence and downregulation of TNF-α, MMP9, and ICAM1. All data are presented as mean ± S.E.M. ns indicated no significance, *P < 0.05, **P < 0.01 (Pearson’s correlation test for B and C; two-tailed Student’s t-test for D–E, H, J, and N).