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. 2012 May 31;8(5):761–777. doi: 10.7150/ijbs.4036

Figure 3.

Figure 3

Physical and functional interaction of ZFP36L1 and ZFP36L2 with 3′UTR of Mkp-1 mRNA (A) RNA pull-down shows the interaction between the TTP family and Mkp-1 mRNA 3′UTR. Biotinylated Mkp-1 3′UTR was incubated with cytosolic extracts from 3T3-L1 cells induced with FMDI for 0, 0.5, 1, and 2 h, and then brought down by streptavidin-Sepharose beads. Biotinylated 18S rRNA served as a negative control. The RNA-protein complexes were separated by SDS-PAGE and immunoblotted with anti-TTP, anti-ZFP36L1, and anti-ZFP36L2. β-tubulin served as a negative control. A representative of three independent experiments with similar results is shown. (B) ZFP36L1 and ZFP36L2 downregulate Mkp-1 3′UTR-driven luciferase activity. HEK293T cells were co-transfected with 0.2 μg or 0.5 μg of FLAG-tagged ZFP36L1 or ZFP36L2 expression plasmid and a pCMV-Luciferase reporter construct containing the Mkp-1 3′UTR. The ARE-derived luciferase assay results were normalized with β-galactosidase activity to correct for variations in transfection efficiency, and also normalized with the activity of a luciferase reporter lacking the ARE to compensate for transcriptional activation (upper panel). The lower panel shows the ectopic expression of the proteins as revealed by immunoblotting with antibodies against the indicated proteins. Each reaction contained duplicate cultures, and each experiment was repeated 3 or 4 times independently. **P < 0.01; ns, not significant. (C) ZFP36L1 and ZFP36L2 downregulate Mkp-1 3′UTR-mediated Luciferase RNA stability. HEK293T cells were co-transfected with pCMV-Luciferase-MKP-1 3′UTR, FLAG-ZFP36L1, or FLAG-ZFP36L2 expression plasmid, and pCMV-β-galactosidase. After 24 h, the cells were left untreated or treated with 10 μg/ml actinomycin D to block transcription for 30 min or 60 min. RNA was subjected to quantitative PCR analysis using primers directed to the luciferase and β-galactosidase genes. The relative Luciferase mRNA levels are shown. The mRNA half-life (t1/2) was calculated and indicated. *P < 0.05, **P < 0.01.