RNA electrophoretic mobility shift analyses of ZFP36L1 binding to the <i>BCL2</i> ARE.

<p>(A) REMSA analysis of recombinant ZFP36L1 binding to <i>BCL2</i> ARE RNA probes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102625#s2" target="_blank">Methods</a>). 50 ng of purified recombinant ZFP36L1 protein was incubated with ³²P-labeled <i>BCL2</i> ARE. The specificity of RNA-protein complexes were examined by addition of 80 ng of anti-ZFP36L1 antibody (Cell Signaling Technology rabbit-anti-human ZFP36L1/L2 antibody) or a non-specific control (rabbit IgG) for 10 min prior to addition of radiolabeled probes as indicated (B) REMSA analysis of recombinant ZFP36L1 binding to <i>BCL2</i> ARE probes, using cold competition. 50 ng of purified recombinant ZFP36L1 protein was incubated with ³²P-<i>BCL2</i> ARE (lane 2) or ZFP36L1 and 5× (lane 3) and 10× molar (lane 4) excess of unlabeled cold competitor <i>BCL2</i> ARE probe. Lane 1: <i>BCL2</i> ARE probe only. (C) REMSA analysis of recombinant ZFP36L1 binding to <i>BCL2</i> ARE probe, using decreasing amounts of recombinant ZFP36L1 protein. Decreasing amounts of the purified ZFP36L1 protein (50–2.5 ng) were incubated with radiolabeled <i>BCL2</i> ARE probe. Lane 1: <i>BCL2</i> ARE probe alone; Lane 2, 3, 4, and 5: <i>BCL2</i> probe and 50, 25, 5, and 2.5 ng of recombinant ZFP36L1 protein respectively. The REMSA experiments described above were repeated at least twice with similar results. (D) Western blot analysis of ZFP36L1/L2 protein expression in non-induced and PMA-induced Ramos cells. Ramos B cells were serum-starved for 16 hours either unstimulated or stimulated with 50 nM PMA for 3 hours Lane 1: Cell lysate from non-induced Ramos cells; Lane 2: Cell lysate from PMA-induced Ramos cells. Anti-human ZFP36L1/L2 antibody (Cell Signaling Technology rabbit-anti- human ZFP36L1/L2 antibody) was used to detect ZFP36L1/L2 expression. An anti-HSP90 antibody was used as a control for protein loading on the gel. The data shown is representative of similar results obtained in two independent experiments. (E) PMA-induced lysates (30 µg) of Ramos B cells were incubated with radiolabeled <i>BCL2</i> ARE and 80 ng of anti-ZFP36L1 antibody (Cell Signaling Technology rabbit-anti- human ZFP36L1/L2 antibody) and non-specific (rabbit IgG) antibodies respectively. Lane 1: <i>BCL2</i> ARE probe alone; Lane 2: <i>BCL2</i> ARE probe incubated with PMA-induced Ramos lysate; Lane 3: <i>BCL2</i> ARE probe incubated with PMA-induced Ramos lysate and anti-ZFP36L1/L2 antibody; Lane 4: <i>BCL2</i> ARE probe incubated with PMA-induced Ramos lysate and non-specific control antibody. * indicates specific bands.