Examination of the binding of PTB-containing protein complexes on p19^Ink4d mRNA.

<p>(A) Ribonucleoprotein immunoprecipitation and RT-qPCR assays. H1299 cells were transfected with either pCMV-SPORT6 (EV) or pCMV-SPORT6-PTB (PTB), and lysed 24 h post-trsnsfection. One milligram of lysates prepared from EV or PTB cells were incubated with protein A beads precoated with 15 µg of either anti-PTB or anti-cyclin A1 (anti-cycA1) antibody to precipitate ribonucleoprotein complexes and to extract RNAs from the complexes as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058227#s2" target="_blank">Materials and Methods</a>. RNAs were used in subsequent RT-qPCR assays for p19^Ink4d and β-actin mRNAs. p19^Ink4d signals were normalized to β-actin signals. The normalized p19^Ink4d signals obtained from the ribonucleoprotein complexes pulled down by anti-PTB antibody were compared with those pulled down by anti-cyclin A1 antibody (to which a value of 1 was assigned). Data represent the means ± S.D. from three independent analyses. (B) Western blot analyses. Forty micrograms of the immunoprecipitates pulled down by anti-PTB antibody-coated beads were subjected to Western blot analyses for the levels of PTB. A representative blot is shown.