Detection of poly(A) interacting proteins using UV crosslinking.

<p>RRL (A), HeLa (B), or Krebs (C) S10 cytoplasmic extracts were subjected to UV-induced crosslinking with the ³²P-poly(A) tail. The extracts that were either mock-depleted (Control S10) or depleted of PABP were incubated with ³²P poly(A) tail-labeled globin mRNA at 32°C for 10 min. Prior to adding mRNA, the reaction mixtures were pre-incubated at 32°C for 2 min with GST-Paip2 (20 µg/ml) or PABP (5 µg/ml) as indicated. After UV irradiation and RNase treatment, labeled proteins were analyzed by SDS-PAGE and autoradiography. The positions of molecular mass markers are indicated on the right. Western blotting with an anti-PABP antibody was used to confirm sufficient depletion of PABP from the extracts (for representative analyses, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001564#pbio.1001564-Svitkin2" target="_blank">[54]</a>). Of note, in this and other UV crosslinking analyses, PABP appeared as a fussy band, most probably because RNase digestion does not produce homogenous cross-linked RNA fragments.