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Figure 1. CRM1-dependent nuclear export activity of E1A. A) Coimmunoprecipitation of E1A and CRM1 from nuclear extract of 293 cells. mIgG, mouse IgG; Input, 2% of nuclear extract proteins used for immunoprecipitation. B) Nuclear accumulation of E1A induced by LMB in 293 cells. Cells were treated with 10 ng/ml LMB for 16 h. Then nuclear and cytoplasmic proteins were extracted and analyzed by immunoblotting (20 µg proteins per lane, left panel). The response of E1B to LMB was used as a positive control for LMB treatment. Lamin B and 
-tubulin were used as loading controls for nuclear and cytoplasmic proteins, respectively. Nuclear/cytoplasmic (N/C) ratio of proteins is shown in the right panel. N/C ratios of LMB-untreated cells were regarded as 1. C) Subcellular localization of EGFP, EGFP-E1A and EGFP-E1AK fusion proteins in U-2 OS cells and response to LMB. 4',6'-diam idino-2-phenylidole stain was used to indicate the position of nucleus. D) CRM1-dependent cytoplasmic localization of EGFP-E1AK. U-2 OS cells were first transfected with 100 nM siRNAs for 24 h and then transfected with the EGFP-E1AK vector for another 24 h. Cells were stained with an anti-CRM1 antibody (Ab) and Texas Red-conjugated secondary Ab. CsiRNA, control siRNA; siCRM1, siRNA for CRM1. E) Down-regulation of CRM1 by siRNA. U-2 OS cells were transfected with 100 nM siRNA or mock transfected. Proteins in cell lysates were analyzed with immunoblotting 48 h after transfection. -tubulin was used as loading control.
