Fig 1: TRBP affects miRNAs production. (A) RISC-associated proteins of 293 T and TRBP-KO cells were detected by Western blotting. (B) Northern blotting analysis of miR-126a-3p and let-7a-5p expression levels in the indicated cells. The relative expression levels of miRNAs are normalized to that of U6 small nuclear RNA and to the miRNA level from the WT 293 T cells. (C) TRBP promotes endogenous let-7a-5p expression. FLAG-TRBP was transfected into TRBP-KO cells for 48 h. Northern blotting and Western blotting were performed to detect let-7a-5p, U6, TRBP, and β-actin expressions in the indicated cells. The relative expression level of let-7a-5p is normalized to that of U6 small nuclear RNA and to the miRNA level from the TRBP-KO cells. (D) TRBP promotes exogenous miR-21 expression. TRBP-KO cells were transfected with a miR-21 expressing plasmid and FLAG-TRBP or FLAG-EGFP for 48 h. Northern blotting and Western blotting were performed to detect expression levels of miR-21, U6, FLAG-tagged proteins and β-actin. The relative expression level of miR-21 is normalized to that of U6 small nuclear RNA and to the miRNA level from the miR-21-transfected TRBP-KO cells.
Fig 2: NS1-TRBP interaction reduces miRNAs production. (A) The expression level of let-7a-5p is downregulated in PR8-infected WT cells. WT 293 T cells and TRBP-KO cells were infected with mock or PR8-WT (MOI = 1) or PR8/3841 (MOI = 1) for 48 h. Northern blotting and Western blotting were performed to detect expression levels of let-7a-5p, U6, NS1, and β-actin. The relative expression level of let-7a-5p is normalized to that of U6 small nuclear RNA and to the miRNA level from the uninfected WT 293 T cells or the uninfected TRBP-KO cells. (B) NS1-TRBP interaction reduces the expression of endogenous let-7a-5p. TRBP-KO cells were transfected with FLAG-TRBP for 24 h before infected with PR8-WT or PR8/3841 for 24 h. Northern blotting detection of let-7a-5p and U6 expressions. The relative expression level of let-7a-5p is normalized to that of U6 small nuclear RNA and to the miRNA level from the uninfected cells. Western blotting detection of TRBP, NS1, and β-actin proteins. (C, D) NS1-TRBP interaction reduces the expression of exogenous miR-21. (C) TRBP-KO cells were transfected with a miR-21 expressing plasmid for 24 h before infected with mock or PR8-WT or PR8/3841 for 24 h. (D) TRBP-KO cells were co-transfected with FLAG-TRBP and miR-21 expressing plasmid for 24 h before infected with mock or PR8-WT or PR8/3841 for 24 h. Northern blotting detection expressions of miR-21 and U6. The relative expression level of miR-21 is normalized to that of U6 small nuclear RNA and to the miRNA level from the uninfected cells. Western blotting detection of TRBP, NS1, and β-actin proteins.
Supplier Page from OriGene Technologies for MIR21 Human MicroRNA Expression Plasmid (MI0000077)