Additional file 1 of C9orf72-associated SMCR8 protein binds in the ubiquitin pathway and with proteins linked with neurological disease
Creators: name: Goodier, John L.
name: Soares, Alisha O.
name: Pereira, Gavin C.
name: DeVine, Lauren R.
name: Sanchez, Laura
name: Cole, Robert N.
name: García-Pérez, Jose Luis
Publisher: name: figshare
Year: 2020
Subjects: subject: Biophysics
subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Immunology
subject: FOS: Clinical medicine
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer
subject: 110309 Infectious Diseases
subject: FOS: Health sciences
subject: FOS: Health sciences

description: Additional file 1 Fig. S1. Western blotting of cell line and brain tissue lysates using commercial antibodies against C9orf72, SMCR8, and WDR41 proteins. Antibodies shown are (A) α-C9ORF72-PT (Proteintech Group 22637–1), (B) α-SMCR8-PT (Proteintech 21125–1-AP, (C) α-SMCR8-B (Bethyl Laboratories A304-694A), (D) α-SMCR8-ab186504 (Abcam ab186504), (E) α-SMCR8-ab202283 (Abcam ab202283), (F) α-WDR41-SC (Santa Cruz sc-137923), and (G) α-WDR41-PT (Proteintech 26817–1-AP). Large arrows mark full-length protein products. The small arrows in (F) and (G) mark the presumed minor 45.5 kD WDR41 isoform. Samples were run on NuPAGE 4–12% Bis-Tris Protein gels (Novex) and Western blotting was performed according to [81, 82]. Molecular weight markers are Novex Sharp Pre-stained Protein Standard. Motor: motor cortex, Occ: occipital cortex, S.C.: spinal cord; sample identifier numbers are also shown. Fig. S2. Pie chart of results of DAVID (Database for Annotation, Visualization and Integrated Discovery, [83]) analyses of KEGG pathways showing selected functional categories for candidate member proteins of the (A) FL-SMCR8 and (B) C9orf72-FL protein interactomes. Percentages of the total number of proteins identified (Tables S1 and S2) for each category are shown within the slices. Fig. S3. Phylogenetic multi-sequence alignment of SMCR8 protein sequences for ten species. Alignments were made with Clustal Omega 1.2.1 (EMBL-EBI) followed by BoxShade 3.2 ( http://sourceforge.net/projects/boxshade ). Pink shading marks amino acid residues identical in at least 8 species, while green includes conservative replacements. Lysine residues predicted by MS sequencing to be ubiquitinated are boxed in blue (see Table S3). Species shown are: Homo sapiens, human; Pan troglodytes, chimpanzee; Canus lupus familiaris, dog; Mus musculus, house mouse; Rattus norvegicus, brown rat; Gallus gallus domesticus, chicken; Danio rerio, zebrafish; Xenopus tropicalis, western clawed frog; Biomphalaria glabrata, freshwater snail; Aplysia californica, California sea hare. Fig. S4. Immunofluorescence microscopy evidence for association of SMCR8 protein with cytoplasmic aggregates. (A) V5-tagged SMCR8 does not significantly enter SGs of NaAsO2-stressed N2A cells. (B) Overexpression of FLAG-tagged C9orf72 (left) or SMCR8 (right) protein does not induce formation of cytoplasmic granules in neuroblastoma cells. (C) V5-tagged exogenously expressed C9orf72 fails to enter SGs in NaAsO2-treated U2OS cells. (D) Endogenous C9orf72 protein speckles observed in unstressed N2A cells are not marked by TIA1, a SG protein. (E) C9orf72 protein detected by the α-C9orf72-PT antibody fails to enter SGs of NaAsO2-stressed U2OS cells. (F,G), Endogenous C9orf72 protein detected by the α-C9orf72-SC antibody colocalizes or justaposes with only a minor subset of SGs (marked by TIA1) or PBs (marked by 4-ET) in stressed mouse neuroblastoma N2A cells (see arrows). (H) In NaAsO2-stressed 293T cells, endogenous SMCR8 detected by the α-SMCR8-PT antibody colocalizes with granules marked by ORF1p, a protein encoded by LINE-1 retrotransposons [81]. (I) In NaAsO2-stressed human neuroblastoma SK-N-SH cells, endogenous SMCR8 localizes to SGs marked by α-eIF3η antibody. (J) In N2A cells treated with the endoplasmic reticulum stressor thapsigarin, endogenous SMCR8 protein colocalizes to granules marked by HEDLS/EDC4 (detected by α-p70 S6 kinase antibody, [77]), a component of PBs. (K) In unstressed N2A cells, endogenous SMCR8 localizes in granules with GW142, a PB marker. (L) Exogenously expressed TDP-43 and endogenous SMCR8 proteins do not colocalize in cytoplasmic granules of stressed U2OS cells. (M) SMCR8 rings but is generally excluded from overexpressed GFP-(GA)50 dipeptide aggregates. (M) In 293T cells, the α-WDR41-PT antibody detects endogenous protein in or beside a subset granules marked by 4-ET (see arrows). NT: no treatment. Cell nuclei were stained with Hoechst 33342 (right-most panels). Size bars are 10 μm. Table S1. The C9orf72 protein interactome determined by mass spectrometry. Table S2. The SMCR8 protein interactome determined by mass spectrometry. Table S3. Ubiquitinated SMCR8 lysine residues determined by MS sequencing, prediction algorithms, and the comparative phylogenetic analyses of Fig. S3.Table S4. Post-mortem brain motor cortex tissue samples used for the analyses of Fig. 5b,c.