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. 2018 Apr 15;29(8):988–1002. doi: 10.1091/mbc.E17-02-0105

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© 2018 Qi, Song, et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.

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FIGURE 7: — Ser-52 and Ser-178 of TTP are the main phosphorylation sites mediating its interaction with Ago2 and ARE-mRNA degradation. (A) 293T cells tranfected with Myc-tagged Ago2 and Flag-tagged TTP-WT or TTP-AA were subjected to anti-Myc immunopurification (IP), and precipitates were detected via immunoblotting with anti-Flag antibody. (B) Coimmunoprecipitation assays performed with extracts derived from 293T cells coexpressing Flag-tagged TTP-AA together with MKK6b(E) or MKK6b(A). Immunoprecipitations (IP) were performed with anti-Flag antibody. Pellet and 5% of input fractions were loaded on the left and the right and subjected to immunoblotting with anti-Flag or anti-Ago2 antibodies as indicated. (C) Results of an experiment similar to that for B, except that the anti-Ago2 antibody was replaced with anti-Dcp1a antibody. (D) HeLa cells were transfected with plasmids expressing either TTP-WT-EGFP or TTP-AA-EGFP. PBs were displayed with anti-Dcp1a antibody (left panel). Graph showing the P-body numbers that are positive for TTP, and the total P-body numbers per cell, in TTP-WT-EGFP and TTP-AA-EGFP transfection conditions (right panel). Error bars represent standard error calculations obtained from averaging the PB number for 50 cells. *p < 0.05; ns, no significance. (E) HeLa cells were transfected with two plasmids, one expressing TTP-AA-EGFP and another expressing MKK6b(E), MKK6b(A), or a corresponding empty vector. PBs were visualized by anti-Dcp1a staining. (F) 293T cells were transfected with the FL-GM-CSF reporter and RL plasmids, together with two plasmids, one expressing HA-tagged TTP-WT, TTP-AA, or Septin1 and another expressing either MKK6b(E) or corresponding empty vector, as indicated. The relative value of FL mRNA was set to 1 for cells transfected with the plasmid expressing HA-tagged Septin1 in each condition. Means and SD from three independent experiments are shown. *p < 0.05; **p < 0.01; ns, no significance. (G) 293T cells were transfected with the indicated siRNAs and then transfected with reporter FL-GM-CSF and RL plasmids, together with HA-tagged TTP-AA or HA-Septin1. The normalized value of FL mRNA level was set to 1 for cells transfected with plasmids HA-Septin1 in each knockdown condition. Means and SD from three independent experiments are shown. **p < 0.01.