His-tagged Impα1 constructs were generated from the vector pcDNA3.1/V5-His-TOPO to express either WT-Impα1 or Impα1 bearing point mutations. IEC-6 cells were transfected with the constructs indicated by using Lipofectamine™, and clones resistant to the selection medium were isolated and screened for Impα1 expression by Western-blot analysis using a specific anti-His-tag antibody. Total, cytoplasmic and nuclear proteins were prepared, and levels of HuR were measured by using a specific antibody. Equal loading in total, cytoplasmic and nuclear proteins was monitored by β-actin, β-tubulin and lamin B respectively. Three experiments were performed and they showed similar results.