Figure 8.
Identification of hypoxia-induced RNA-binding proteins using EMSA/UV-cross-linking and RNA affinity chromatography. (A) Normoxic (N) and hypoxic (H) M21 cell extracts bound to the 3′ HSR probe were subjected to UV-cross-linking/RNase treatment and analyzed by reducing SDS-PAGE. Hypoxia-induced proteins covalently cross-linked to the 3′ HSR probe are indicated by arrows. (B) Competitive inhibition of RNA–protein complex formation by excess cold VPF/VEGF 3′ HSR. M21 24-h hypoxic cell extracts EMSA/UV cross-linked to VPF/VEGF 3′ HSR mRNA (−) was competed with unlabeled VEGF 3′ HSR (0.05, 0.5, and 1 μg 3′ HSR, lanes 2–4, respectively). The arrows indicate 3′ HSR mRNA protein complexes.