Figure 3. Ectopically-expressed ELAVL2° acts as a translational repressor (A) Tethering assay principle. A Renilla luciferase reporter carrying 5 BoxB binding sites (RL-5BoxB) interacts with the λN-peptide fused to a tested protein. (B) NHA-ELAVL2° specifically interacts with RL-5BoxB. After NIH 3T3 cell transfection with constructs expressing RL-5BoxB, FL, HA tag, and indicated ELAVL2° proteins, lysates were immunoprecipitated with IgG or α-HA antibodies. mRNA was recovered from pellets and selected transcripts were detected by RT-PCR. The data are normalized to the IgG background (mean ± s.e.m., n = 2). Expression levels (INPUT) and immunoprecipitation efficiency (IP, HA) of tagged ELAVL2° proteins were determined by western blot using α-HA antibody. Tubulin served as a loading control. (C) Tethering assay showing 50% reduction of RL-5BoxB signal in presence of NHA-ELAVL2°. HeLa and NIH 3T3 cells were co-transfected with RL-5BoxB, non-targeted firefly luciferase (FL), and HA-tagged ELAVL2° without (HA-ELAVL2°) or with (NHA-ELAVL2°) λN-peptide. The graph represents relative Renilla luciferase activity (R.L.A.) normalized to FL (mean ± s.e.m, n = 4). Expression of tagged ELAVL2° proteins was monitored by western blot (shown below graphs). Tubulin served as a loading control. **Marks non-specific bands in HeLa cell extract. (D) RT-PCR analysis of RL-5BoxB, FL, and Hprt1 mRNAs in cells expressing differently tagged ELAVL2° proteins. Data represent relative mRNA expression normalized to globin mRNA (mean ± s.e.m, n = 2). (F) Luciferase assays in HeLa cells expressing RL, RL-5BoxB or RL-3′Ctcf reporters, non-targeted FL reporter, and HA tag alone, or indicated ELAVL2° proteins. The graph presents relative Renilla activity normalized to FL (R.L.A., mean ± s.e.m, n = 2). The average RL-5BoxB expression in the presence of the HA tag was set to one (n, independent experiment performed in triplicates).