Fig. 7.

RACK1 knockdown not only reduces cytoplasmic aggregation but also increases nuclear localization of TDP-43^ΔNLS in HEK293T cells. a Representative images showing that in contrast to control cells where distinctive aggregates of HA-tagged TDP-43^ΔNLS are associated with RACK1 co-aggregation in the cytoplasm (arrows), RACK1 siRNA KD not only alleviates cytoplasmic aggregation (white arrowheads) but also leads to nuclear localization (yellow arrowheads) of TDP-43^ΔNLS in a sub-population of transfected cells. Nuclei stained with DAPI in merged images. Scale bar: 10 μm. b Quantification of the average aggregate size in each individual TDP-43^ΔNLS transfected cells shows a decrease following RACK1 KD. c Quantification of TDP-43^ΔNLS nuclear localization from 3 biological repeats shows an increase as a result of RACK1 KD. d Western blot analysis confirms the quality of nucleocytoplasmic fractionation using nuclear and cytosolic protein markers, lamin B1 and β-Actin, respectively (top). TDP-43^ΔNLS (HA) or total TDP-43 (pan TDP-43) from either fraction was normalized to β-actin or lamin B1. Quantification by densitometry shows that RACK1 KD decreases the cytoplasmic to nuclear ratio, fragmentation (red arrows, observed in 3 independent experiments), and phosphorylation of TDP-43^ΔNLS (quantitated in one experiment). Statistics: Student’s t-test unpaired two-tailed. Error bars: SEM