Fig. 5
From: A complex of C9ORF72 and p62 uses arginine methylation to eliminate stress granules by autophagy
p62 regulates turnover of FUS and splicing of its targets. a Western blot of symmetrically dimethylated arginines in FUS immunoprecipitates from HEK293T cells. b Left, western blot of FUS immunoprecipitates from HeLa cells treated with arsenite and Bafilomycin A1; Right, western blot of FUS in HeLa cells in conditions used for PLA in (c). c Left, PLA of p62 and wild type or indicated FUS mutants in HeLa cells treated with arsenite and Bafilomycin A1; Right, quantification of PLA; images counted (>10 cells/image): wild type (11), R218K (9), R216C (8), R216C + R217K (8), R521G (9), R521H (9) (n = 3, mean ± SD, Student’s t-test). d Quantification of nuclear/cytoplasmic distribution of wild type and mutant FUS by fluorescence intensity in HeLa cells treated with arsenite and Bafilomycin A1; cells counted: wild type (32), R218K (19), R216C (8), R216C + R218K (16), R521G (31), R521H (21) (mean ± SD, Student’s t-test). e Left, western blot of FUS wild type, indicated mutants and LC3 upon Bafilomycin A1 treatment of HeLa cells; Right, quantification of western blots normalized to Tubulin (n = 6, mean ± SD, Student’s t-test). f Left, percentage of cells containing SGs in primary motor neuron cultures of wild type and p62−/− mice (n = 2–4); 35–150 cells were counted per condition (mean ± SD, Student’s t-test); Right, representative images. g Western blot of lysates from wild type (WT) and p62−/− (KO) mice. h–l RT-PCR analysis of Zcchc6 splice variants in (h) p62 depleted MN1 cells (n = 4), i indicated tissues of WT and p62−/− mice (n = 3), j FUS depleted and overexpressing MN1 cells (n = 4), k FUS and p62 depleted MN1 cells (n = 3), and l MN1 cells depleted of endogenous FUS with siRNA targeting FUS 3’UTR and expressing FUS wild type or indicated mutants (n = 4, mean ± SEM, Student’s t-test). m Venn-diagram depicting overlap between mRNAs putatively alternatively spliced in p62-/- brains and those bound to FUS and mis-spliced in FUS-/- brains. n A plot of the fraction of mRNAs putatively mis-spliced in p62−/− brain (y-axis) vs. the increasing significance of this (-log10 p-value, x-axis); black line—all detected mRNAs, green line—mRNAs enriched in FUS-binding motifs, red line—only mRNAs putatively mis-spliced in p62−/− brain. p = 0.0357 that putative mRNAs mis-spliced in p62−/− brains are enriched in FUS-binding motifs. Scale bar = 10 µm
