Fig. 1
From: A complex of C9ORF72 and p62 uses arginine methylation to eliminate stress granules by autophagy
C9ORF72 associates with ALS-linked stress granule proteins including p62. a Western blot of endogenous C9ORF72 immunoprecipitates compared to immunoprecipitation with non-specific IgG from cell lysates (input) transfected with control or C9ORF72 siRNA. Secondary antibody specific for IgG light chain was used. b Western blot of endogenous C9ORF72 immunoprecipitates with and without arsenite treatment. c Left, Venn-diagram depicting the number of proteins in the putative C9ORF72 interactome that localize to stress granules (SGs, blue) or contain mutations linked to ALS (olive); Right, western blot of immunoprecipitation of C9ORF72-myc for p62 and SG proteins (FUS, SMN). d Immunofluorescence of endogenous C9ORF72 and SGs (FMRP) after 30 min incubation with Sodium Arsenite, (0.5 mM); Right, quantification of the percentage of SGs (FMRP+) co-localizing with foci of C9ORF72 (quantified 3916 C9ORF72 punctae and 3331 SGs). e Western blot of HA-p62 immunoprecipitates with and without arsenite treatment. f PLA co-labeling with species controlled IgG (top, negative control) and known interactors p62 and LC3 (bottom, positive control); p62-LC3 association was tested in cells treated with Bafilomycin A1 (Baf A1). g Top, proximity ligation assay (PLA) for C9ORF72 and NBR1 in arsenite-treated cells; Bottom, immunofluorescence for NBR1 showing antibody does label cells. h PLA for endogenous p62 and C9ORF72 in cells treated with arsenite and transfected with control (Ctl) or C9ORF72 (C9) siRNA. i Quantification of PLA signals for C9ORF72 and p62 in the cytoplasm and nucleus in identical conditions to (h) (n = 3, mean ± SD, Student’s t-test). j Western blot of recombinant p62 and GST-C9ORF72 from in vitro pulldown assay. k Representative immunoprecipitation of HA-p62 used for identification of putative p62 interacting proteins using LC-MS/MS. l Venn-diagram depicting number of proteins in predicted p62 interactome that localize to SGs (blue). m Western blot of immunoprecipitates of HA-p62 detecting known interactors (LC3, ubiquitinated proteins), candidate interactors identified by LC-MS/MS (GFP-DDX3) and control RNA-binding proteins that do not associate with p62 (EWS) in cells treated or not with arsenite. n Western blot of immunoprecipitations of endogenous p62 (top) and HA-p62 (bottom) for other SG proteins (FUS, SMN). HeLa cells were used in all experiments. Scale bar = 10 µm. Molecular weight markers for blots, where not shown, are included in the uncropped versions
