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. 2016 Sep 15;30(18):2076–2092. doi: 10.1101/gad.282616.116

Figure 6.

Figure 6.

DUSP11 promotes accumulation, AGO association, and RISC activity of AdV5 mivaRNAI-5p. (A) Northern blot analysis of the 5p and 3p miRNAs derived from AdV5 VA RNAI from parental (wild-type [WT]), DUSP11 knockout (D11-KO), DUSP11-KO-pLenti-D11, and DUSP11-KO-pLenti-DUSP11-CM HEK293T cells transfected with pVAI. The membrane was first probed for the 5p miRNA, stripped, and reprobed for the 3p miRNA. (B) AGO RIP analysis of VAI-derived miRNAs. Parental (wild-type) and DUSP11 knockout HEK293T cells were cotransfected with pcDNA-EV, pAGO1-Flag, or pAGO2-Flag and either cotransfected with pVAI or infected with AdV5. RIP was performed 48 h after transfection using anti-Flag M2 antibody. One percent of the input RNA and 95% of RIP-recovered RNA were subjected to Northern blot analysis. The membrane was first probed for the 5p miRNA, stripped, and reprobed for the 3p miRNA. Immunoblot analysis was performed on 0.1% of input lysate and 5% of the Flag RIP. (C) Luciferase assay to measure RISC activity of the 5p miRNA derived from VAI RNA in parental (wild-type), DUSP11 knockout, DUSP11-KO-pLenti-D11, and DUSP11-KO-pLenti-DUSP11-CM HEK293T cells. Bars represent the mean luciferase ratio (Renilla/firefly) ± SEM from three experiments (wild-type and DUSP11 knockout) or four experiments (stable cell lines) in which transfections were performed in triplicates.