RNA Binding Activity Assay.

<p>TNF mRNA ARE binding activity of the wild-type and mutant TTP was evaluated with the RNA gel mobility shift assay. (A) Electrophoretic mobility shift assay. The binding reaction mixtures were incubated for 30 min followed by RNase T₁ digestion. The TTP−probe complexes and free probes were separated by 6% native PAGE and detected by autoradiography on X-ray film. (B) TNF mRNA ARE probe. The RNA probe was transcribed from mouse TNF mRNA ARE region with the following sequence: 5′-CUCUAUUUAUAUUUG|CACUUA<u>UUAUUUA<b>UU</b></u><b>AU<u>UUAUUUAUU</u>AU<u>UUAUUUAUU</u></b>UG|CUUAUG|AAUG|UAUUUA-3′ (the three UUAUUUAUU repeats are underlined, and the two overlapping repeats (<b>UUAUUUAUUUA<u>UU</u>AUUUAUUUAUU</b>) are in bold letters; the vertical bar represents the RNase T1 recognition site).