Fig. 6

Validation of RBPs using PNK assay. Briefly, 3Flag-HBH-tagged proteins are stably expressed in Flp-In T-Rex 293 cells. The proteins are affinity purified from UV crosslinked and non-crosslinked cells under denaturing conditions. RNA bound to proteins is labelled with radioactive ³²P using the PNK enzyme. The RNA–protein heteroconjugates are separated by size (SDS-PAGE) and transferred to a membrane on which the RNA binding capacity is detected by autoradiography (RNA band labelled with red asterisk). The proteins are detected by western blot (bottom) using anti-Flag antibody (protein band labelled with green asterisk). The assay was performed on the following class of proteins: a RNA-related proteins comprising the classical RRM domain-containing RBPs serrate RNA effector molecule (SRRT) and peptidylprolyl isomerase E (PPIE), as well as WD40 domain-containing protein receptor for activated C kinase 1 (RACK1) and splicing related protein Sin3A-associated protein 18 (SAP18). b Cell signalling proteins phosphatidylethanolamine binding protein 1 (PEBP1) and casein kinase 1 delta (CSNK1D). For PEBP1 an additional control with high amounts of RNase I treatment (100 units) is included alongside the standard (1 unit) treatment employed in all experiments. c Cytoskeleton ERM family proteins Radixin (RDX), Ezrin (EZR) and Moesin (MSN). An additional control with high amounts of RNase I is included for RDX. d Metabolic enzymes aldehyde dehydrogenase 6 family member A1 (ALDH6A1), malonyl-CoA-acyl carrier protein transacylase (MCAT) and short/branched chain acyl-CoA dehydrogenase (ACADSB)