Figure 4.

HuR interaction with poly(A)⁺ RNA shifts from the cytoplasm to the nucleus after heat shock. HeLa (5 × 10⁶) cells, grown at 37°C (lanes 2, 3, 6, and 7) or incubated for 1 h at 45°C (lanes 4, 5, 8, and 9) were exposed to Stratalinker UV for 15 min to induce covalent RNA–protein crosslinks in vivo. Poly(A)⁺ RNA-protein crosslinked complexes were then isolated from the cytoplasmic (C) and nuclear (N) fractions by chromatography on oligo(dT) in the presence of 1% SDS and β-mercaptoethanol (28) to strip away noncovalently bound proteins; the absence of signal in lanes 2–5 shows this strategy to be successful. Crosslinked proteins were released by digestion with RNase, resolved by SDS/PAGE; and HuR, hnRNP A1 and hnRNP D were detected by sequential probing of the same blot with the respective antibodies. In lanes 2–5, the cells were treated exactly the same, but the UV treatment was omitted. Lane 1 shows the proteins present in 1/100 of the 37°C UV-treated extract before separation into the nuclear and cytoplasmic fractions. These experiments were reproduced three times.