Functional association of TTP with <i>PIM1</i> mRNA in HeLa cells.

<p>(A) RNP-IP experiments were performed using control IgG or anti-FLAG antibodies and lysates from untransfected HeLa/Tet-Off cells (ut) or stable clonal lines expressing FLAG-TTPwt or FLAG-TTP C147R as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033194#s2" target="_blank">Materials and Methods</a>”. Immunoprecipitated material was then screened for <i>PIM1</i> mRNA by quantitative real-time RT-PCR and normalized to GAPDH mRNA (mean ± SD of three reactions). Independent replicate experiments yielded similar results. (B) Relative levels of <i>PIM1</i> mRNA were measured in untransfected <i>versus</i> FLAG-TTPwt- or FLAG-TTP C147R-expressing HeLa cells. Bars represent the mean ± SD of quadruplicate qRT-PCR reactions normalized to GAPDH mRNA. (C) The decay kinetics of <i>PIM1</i> mRNA was measured in HeLa cell models using actD time course assays as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033194#pone-0033194-g002" target="_blank">Figure 2</a>. mRNA half-lives calculated from independent replicate experiments are provided in the text. (D) Western blot analyses using antibodies targeting specified proteins in HeLa/Tet-Off cell models, with positions of molecular weight markers (in kDa) shown at left.