FIG. 6.

In vitro analysis of interactions between ectopically expressed human HuR and its mutant derivatives with the c-fos ARE. ³²P-labeled ARE RNA transcribed in vitro from a T3 promoter-driven template was incubated with cytoplasmic lysates from NIH 3T3 B₂A₂ cells expressing no ectopic protein (vector), Myc-tagged HuR (wild type [WT]), or a mutant protein (ΔI, ΔB, or ΔIII). The RNA-protein complexes were then digested with RNase T₁. The digestion mixtures were left alone (−) or further incubated in the presence of the 9E10 monoclonal antibody (MAb) against the Myc epitope tag. The final reaction mixtures were resolved by electrophoresis in a native polyacrylamide gel. Antibody-supershifted complexes and free probe (FP) are indicated.