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. 2017 Feb 28;292(14):5784–5800. doi: 10.1074/jbc.M116.763599

Figure 3.

Figure 3.

eEF1A1 is involved in the cytoplasmic mislocalization of expanded poly(A) tract-containing nuclear exporter reporter protein. A, subcellular localization of Rev(1.4)-poly(A)-EGFP protein in HEK293 cells after eEF1A1 knockdown. The knockdown of eEF1A1 expression caused a nuclear enrichment of Rev(1.4)-A37-EGFP protein but had no effect on the subcellular localization of Rev(1.4)-A6-EGFP protein. The cell nuclei were stained with Hoechst 33432. Scale bar, 10 μm. B, statistical analysis of A. Knockdown of eEF1A1 expression caused a statistically significant increase in the percentage of cells expressing Rev(1.4)-A37-EGFP protein with an N/C ratio of >1. C, the knockdown efficiency of eEF1A1 was measured using immunoblotting. β-Tubulin served as the loading control. D, subcellular localization of Rev(1.4)-poly(A)-EGFP protein in HEK293 cells after Myc-eEF1A1 or Myc-eEF1A1Δ429–449 overexpression. Overexpression of Myc-eEF1A1 caused Rev(1.4)-A37-EGFP protein cytoplasmic enrichment but had no effect on the localization of Rev(1.4)-A6-EGFP protein. Overexpression of Myc-eEF1A1Δ429–449 had no effect on the localization of both Rev(1.4)-A6-EGFP and Rev(1.4)-A37-EGFP. The cell nuclei were stained with Hoechst. Scale bar, 10 μm. E, statistical analysis of D. Overexpression of Myc-eEF1A1 caused a statistical significant decrease in the percentage of cells expressing Rev(1.4)-A37-EGFP protein with N/C ratio >1. F, Myc-eEF1A1 overexpression detection using immunoblotting. β-Tubulin served as the loading control. G, subcellular localization of Rev(1.4)-poly(A)-EGFP protein in HEK293 cells after eEF2 knockdown. The knockdown of eEF2 expression had no effect on the subcellular localization of both Rev(1.4)-A6-EGFP and Rev(1.4)-A37-EGFP protein. The cell nuclei were stained with Hoechst. Scale bar, 10 μm. H, statistical analysis of G. Knockdown of eEF2 expression did not change the percentage of cells expressing Rev(1.4)-poly(A)-EGFP protein with N/C ratio >1. I, the knockdown efficiency of eEF2 was measured using immunoblotting. β-Tubulin served as the loading control. Three independent transfection experiments were performed. At least 100 transfected cells were measured per experiment. Error bars, S.E. **, p < 0.01. ns, not statistically significant.