FIGURE 4.

Affinity chromatography identification of RBPs interacting with the ASCL1 mRNA UTRs. Kelly neuroblastoma cells were subjected to control or hypoxic conditions for 24 h. Cellular extracts were prepared and used for affinity chromatography with in vitro transcribed biotinylated transcripts representing the ASCL1 mRNA 5′- or 3′-UTR as bait. a, representative silver-stained gel of the captured proteins binding to either the ASCL1 mRNA 5′- or 3′-UTR under control or hypoxic conditions. A no-transcript sample served as a negative control (beads only). b, Western blot confirmation of the trans-acting factors identified by MALDI/TOF-MS analysis (see Table 1). Cellular extracts that were used for affinity chromatography are shown in the input lanes. hnRNP-K and tubulin served as negative controls.