ALS-associated FUS mutants are deficient in alternative splicing and autoregulation.

<p>A) Confocal fluorescent microscopy showing the cellular localization of EGFP-FUS mutants in HEK293 cells. Magnification, 40×. Scale bar, 20 µm. Endogenous FUS protein was detected using anti-FUS antibody (Bethyl, BL1355). DNA in the nucleus was stained with DAPI or NucRed Dead 647. B) RT-PCR analysis of <i>FUS</i> exon 7 splice variants in pDUP-FUS-E7L coexpressed with either wildtype (wt) or mutant EGFP-FUS in HEK293 cells. Bar graphs represent mean ± SD (n = 3). EGFP-FUS mutants were compared with EGFP-FUS wildtype protein using student's <i>t</i>-tests. C) Western blot analysis of endogenous FUS and EGFP-FUS protein in HEK293 cells, using anti-FUS antibody (10F7). Bar graphs represent mean ± SEM (n = 3). EGFP, EGFP-FUS wildtype and mutants were compared with mock transfected cells using student's <i>t</i>-tests. In both panel B and C, * <i>P</i>≤0.05, ** <i>P</i>≤0.01. D) Confocal fluorescent microscopy showing the localization of endogenous FUS protein in the cytoplasmic aggregates of EGFP-FUS ΔE15 mutants expressed in HEK293 cells. Anti-FUS antibody (Bethyl, BL1355) recognizing C-terminus epitope detected only endogenous FUS protein but not ΔE15 mutants. Magnification, 40×. Scale bar, 20 µm.