Fig. 5

RACK1 knockdown alleviates aggregation and restores global translational suppression by FUS^ΔNLS mutants. a Representative SUnSET-Western Blot (WB) demonstrates that compared to control empty vector (EV) transfected cells, expression of HA-tagged FUS^ΔNLS mutants, R495x- and P525L-FUS, and to a lesser extent over-expression of WT FUS, induces a significant reduction in puromycin (PMY) incorporation. b siRNA KD alleviates global translational levels in all cases as determined by quantification of PMY band intensities normalized to loading control α-tubulin. c SUnSET-ICC demonstrates HA-tagged FUS^ΔNLS mutants, R495x- and P525L-FUS, cause aggregation and completely inhibit PMY incorporation in transfected cells (asterisks). d RACK1 KD alleviates aggregation, resulting in diffuse cytoplasmic expression or predominantly nuclear localization of HA-tagged FUS^ΔNLS mutants, R495x- and P525L-FUS, in a sub-population of transfected cells and correlating with normal PMY incorporation compared to neighbouring HA-negative UT cells. Nuclei stained with DAPI in merged images. Statistics: Student’s t-test, unpaired, two-tailed. *p < 0.05 and **p < 0.005. n = 5 Error bars: SEM. Scale bars: 20 μm