CREST co-aggregates with FUS but not with TDP-43 or TAF15. (A) Endogenous FUS co-immunoprecipitates with CREST-GFP. Pull-down of GFP-tagged CREST from transfected cells was performed with GFP-Trap beads as described in Material and methods, endogenous FUS was detected in the immunoprecipitate (IP) by Western blotting (WB). (B) CREST-Flag recruits GFP-tagged FUS (top panel) and its cytoplasmically mislocalised variant bearing R522 substitution (FUS R522G, middle panel) into nuclear aggregates upon co-expression in SH-SY5Y cells. In a small fraction of co-expressing cells the latter variant also co-aggregates with CREST in the cytoplasm (middle, arrowheads, border of the nucleus is indicated by a dashed line in the inset) but in the majority of such cells it is trapped in the nucleus leading to its lowered cytoplasmic levels and significant decrease in the proportion of cells bearing cytoplasmic FUS aggregates (FAs, bottom panel). The number of cells with aggregates was quantified for cells expressing GFP-tagged FUS R522G only (FUS-GFP) and those co-expressing GFP-tagged FUS R522G and CREST-Flag (FUS + CREST). The bar chart shows the fraction of cells (mean ± s.e.m.) bearing FAs (***p < 0.001; at least 100 cells counted per variant from three independent experiments). (C) CREST-GFP and FUS-Flag with R522G substitution co-aggregate. (D) N-terminally truncated protein (FUS-GFP CT, aa. 360-526) cannot be recruited to CREST-Flag aggregates (top panel), while C-terminally truncated FUS (FUS-GFP NT, aa.1-359) retains the ability to co-aggregate with CREST (bottom panel). (E) CREST does not sequester wild-type TDP-43 into nuclear aggregates and is not recruited to cytoplasmic (arrow) or nuclear (arrowheads) aggregates formed by mislocalised TDP-43 (TDP-43-GFP dNLS) or C-terminal TDP-43 fragment (TDP-43-GFP CT, aa.193-414). NLS of TDP-43 was deleted to achieve cytoplasmic re-distribution and aggregation of the protein. (F) Nuclear aggregates of Flag-tagged CREST and GFP-tagged TAF15 do not overlap. Scale bars, 10 μm.