Fig. 4. Identification of proteins bound to the αENaC 3'UTR by RNA affinity chromatography. RNA-binding proteins (RBP) present in extracts of alveolar epithelial cells were purified by affinity chromatography using in vitro transcribed αENaC 3'UTR. Nonspecific RNA binding was tested with an irrelevant RNA (Irr. RNA) consisting of the negative strand of β-actin ORF. (A) The affinity-purified RBPs were separated by SDS-PAGE, and silver-stained bands were subjected to LC-MS/MS for protein identification. A representative silver-stain gel from three experiments from different rats is shown. (B) The RBPs identified by mass spectrometry were confirmed using immunoblotting. The affinity-purified RBPs were separated by SDS-PAGE followed by immunoblotting against Dhx36, hnRNPK or Tial1, showing enrichment of these proteins with αENaC 3'UTR compared to irrelevant RNA. Representative immunoblots for each RBP are presented (original blot shown in Fig. S5). (C) The enrichment of each RBP determined by immunoblot analysis are depicted for each RBP. The data are expressed as relative enrichment ± SEM compared to irrelevant RNA. *P<0.05 by one-sample t-test compared to irrelevant RNA (n=4 for each RBP).