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. 2013 Apr 3;110(19):7778–7783. doi: 10.1073/pnas.1219643110

Fig. 4.

Fig. 4.

Pur α binds rGGGGCC repeats in a dose-dependent manner in vitro and in vivo. (A) rGGGGCC binding assay with mouse and Drosophila Pur α and mouse hnRNP A2/B1. 32P-labeled r(GGGGCC)10 was incubated with increasing concentrations of recombinant Pur α (mouse), Pur α (Drosophila), hnRNP A2/B1, and GST alone. (B) In vivo interaction of rGGGGCC repeats and Pur α. Neuro-2a cells were cotransfected with the FLAG-tagged Pur α and either EGPF, (GGGGCC)3-EGFP, or (GGGGCC)30-EGFP constructs, and immunoprecipitation was performed with either mouse IgG or anti–FLAG-M2 antibodies. (Upper) Western blots of precipitate using IgG control and anti–FLAG-M2 antibodies. The input lane is a Neuro-2a cell expressing FLAG-tagged Pur α alone. (Lower) Results of qRT-PCR for EGFP RNA for each precipitate. (C) Pur α binds rGGGGCC from mouse or human brain lysate. Biotinylated (GGGGCC)10 repeats were incubated with brain lysate. Lanes 1 and 5, 30 µg of brain lysate; lanes 2 and 6, 300 µg of brain lysate; lanes 3 and 7, 300 µg of brain lysate incubated with 300 µM biotin; lanes 4 and 8, 300 µg brain lysate incubated with 300 µM biotinylated r(GGGGCC)10 repeat.