1. Volume 22, Issue 6, Pages 1473-1483 (February 2018)
Citrullination of RGG Motifs in FET Proteins by PAD4 Regulates Protein Aggregation and ALS Susceptibility 
Chizu Tanikawa, Koji Ueda, Akari Suzuki, Aritoshi Iida, Ryoichi Nakamura, Naoki Atsuta, Genki Tohnai, Gen Sobue, Naomi Saichi, Yukihide Momozawa, Yoichiro Kamatani, Michiaki Kubo, Kazuhiko Yamamoto, Yusuke Nakamura, Koichi Matsuda 
Cell Reports 
Volume 22, Issue 6, Pages (February 2018)
DOI: /j.celrep
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2. Cell Reports 2018 22, 1473-1483DOI: (10.1016/j.celrep.2018.01.031)
Copyright © 2018 The Author(s) Terms and Conditions

3. Figure 1
Mapping of Citrullinated Arginine Residues in PAD4-Expressing Cells
(A) Schematic of the design of the proteomics experiments. HEK293T cells were transfected with a PAD4-expressing or mutant PAD4-expressing vector or the control vector. Trypsinized peptides were analyzed using mass spectrometry.
(B) Western blot assays confirmed the presence of protein citrullination in the PAD4-expressing cells. Anti-modified citrulline is an antibody against a chemically modified form of citrulline.
(C) All of the identified citrullinated proteins were annotated according to GO terms. Categories were scored by a combination of the enrichment ratio and the p value.
(D) Connectivity graph of gene ontologies of the proteins citrullinated by PAD4.
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4. Figure 2 Citrullination Consensus Motif
(A) WebLogo representation of the identified citrullination sites. Amino acid types are indicated by the following colors: green, polar (G, S, T, Y, C, Q, and N); blue, basic (K, R, and H); red, acidic (D and E); and black, hydrophobic (A, V, L, I, P, W, F, and M).
(B) Peptides containing 3 RGG repeats were incubated with PAD4 or mutant PAD4 proteins. White or black arrowheads indicate the peak of RGG peptides with or without citrullination, respectively.
(C) Western blot analysis of whole-cell lysates from PAD4-expressing or mutant PAD4-expressing HEK293T cells using antibodies against citrullinated RGG motifs.
(D) Western blot analysis of whole-cell lysates from U2OS cells treated with Adriamycin (ADR) and a calcium ionophore (A23187) (left and middle). The siRNA constructs were transfected 24 hr prior to ADR treatment (middle panel). A23187 was added 15 min before the cells were harvested. Western blot analysis of whole-cell lysates from HL60 cells treated with DMSO and A23187 is shown (right).
(E) Western blot analysis of immunoprecipitated RGG-containing proteins, including hnRNPA1, EWS, TAF15, RBMX, and DDX17, using the antibody against citrullinated RGG motifs. w, wild-type PAD4; m, mutant PAD4.
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5. Figure 3 Effect of Citrullination on Methylation
(A) Western blot analysis of immunoprecipitated TAF15 (upper) and EWS (lower) using the antibody against modified citrulline. w, wild-type PAD4; m, mutant PAD4.
(B) The proportion of methylation at arginine residues of TAF15 (upper) and EWS (lower) in peptide fragments immunopurified from PAD4-expressing or mutant PAD4-expressing HEK293T cells.
(C) Western blot analysis of immunopurified TAF15 (upper) and FUS (lower) proteins using SMN antibody. w, wild-type PAD4; m, mutant PAD4.
(D) Western blot analysis of immunopurified SMN protein using antibodies against RGG-containing proteins, including TAF15, FUS, EWS, and hnRNPA1. w, wild-type PAD4; m, mutant PAD4.
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6. Figure 4 Effect of Citrullination on Protein Solubility
(A) Soluble and insoluble fractions from HeLa cells that were stably expressing wild-type or mutant TAF15 protein and were transfected with the PAD4-expressing or mutant PAD4-expressing vector were analyzed using western blot assays with anti-HA.
(B) Western blot analysis of immunopurified TDP43 protein using antibodies against TAF15, hnRNPA1, DDX17, and hnRNPK. w, wild-type PAD4; m, mutant PAD4.
(C) Representative images of sodium arsenite-treated MEF cells stained for TAF15 and FUS. Scale bars, 20 μm. The proportion of FUS/TAF15-positive granules in Padi4−/− or Padi4+/+ MEF cells is indicated in the right panel. Data are displayed as mean ± SD of six independent fields from two MEFs (Student’s t test, p =  ).
(D) Soluble fractions from Padi4−/− or Padi4+/+ MEF cells treated with b-isox were analyzed using western blot analysis with antibodies against FUS and hnRNPA1.
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7. Figure 5 Role of PAD4 in Neurodegenerative Disease
(A) eQTL analysis for rs The fold change of PADI4 expression between alleles is shown. The reference allele is C. The p values by t test are shown on the right side. FDR threshold is 0.05.
(B) A hypothetical model regarding RGG modifications and protein aggregability.
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