Journal: The Journal of Biological Chemistry
Article Title: Expanded polyalanine tracts function as nuclear export signals and promote protein mislocalization via eEF1A1 factor
doi: 10.1074/jbc.M116.763599
Figure Lengend Snippet: Knockdown of Ef1a48D expression attenuated the expanded poly(A) SoxN toxicity by mediating its mislocalization. A, the effect of knockdown of eEF1A1 on poly(A)-SoxN-EGFP localization in SK-N-MC cells. Knockdown of eEF1A1 expression caused a nuclear enrichment of A34-SoxN-EGFP protein but had no effect on the subcellular localization of A12-SoxN-EGFP protein. The cell nuclei were stained with Hoechst 33342. Confocal images were acquired and processed using Olympus Fluoview FV1000 version 3.0.2.0 software. Red and green lines were drawn successively across individual cells in the confocal image, with matching plots on the right reporting fluorescence intensity. Scale bar, 10 μm. B, representative blots of the effect of eEF1A1 knockdown on the nuclear and cytoplasmic poly(A)-SoxN protein level. Histone (H3) was used as the nuclear marker. β-Tubulin served as the loading control. C, effect of Ef1a48D knockdown on poly(A)-SoxN toxicity. A34-SoxN expression caused a rough eye phenotype, whereas A12-SoxN and GAL4 driver-alone control did not. Knockdown of Ef1a48D rescued the eye phenotype induced by A34-SoxN. The images of external eyes were taken from 2 days post-eclosion flies. D, transcript levels of poly(A)-SoxN and Ef1a48D. The transcript levels of A12-SoxN and A34-SoxN and the knockdown efficiency of Ef1a48D in flies were detected using RT-PCR at 2 days post-eclosion. The flies were raised at 28 °C. The flies were of genotypes w; gmr-GAL4/+; +/+, w; gmr-GAL4/+; UAS-A12-SoxN/+, w; gmr-GAL4/+; UAS-A34-SoxN/+, w; gmr-GAL4/+; UAS-Ef1a48D-dsRNA/+, w; gmr-GAL4/+; UAS-A12-SoxN/UAS-Ef1a48D-dsRNA, and w; gmr-GAL4/+; UAS-A34-SoxN/UAS-Ef1a48D-dsRNA. Three independent experiments were performed.
Article Snippet: The preactivation, photoactivation, and postactivation steps were controlled automatically by the time controller module of Olympus Fluoview version 2.0c software.
Techniques: Expressing, Staining, Software, Fluorescence, Marker, Reverse Transcription Polymerase Chain Reaction
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