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Additional file 2: Figure S1. Citrullinated proteins accumulate as foci in reactive astroglia (dotted circles) in early disease stages and become widespread in late disease stages in ALS mouse models. (A, B) Double immunofluorescence staining for GFAP and citrullinated protein in ventral horn spinal cords of SOD1G93A and PFN1C71G mice, respectively. Figure S2. PC is not increased in microglia in the spinal cord of ALS mouse models. (A, B) Double immunofluorescence staining for IBA1 and citrullinated proteins in the ventral horn gray matter of SOD1G93A and PFN1C71G mice, respectively. (C, D) Double immunofluorescence staining for IBA1 and citrullinated proteins in the ventral lateral white matter of SOD1G93A and PFN1C71G mice, respectively. The ages of nTg mice are as described in Fig. 2. Figure S3. PCs are not increased in oligodendrocytes in the spinal cord of ALS mouse models. (A, B) Double immunofluorescence staining for Olig2 and citrullinated proteins in ventral horn gray matter of SOD1G93A and PFN1C71G mice, respectively. (C, D) Double immunofluorescence staining for Olig2 and citrullinated proteins in the ventral lateral white matter of SOD1G93A and PFN1C71G mice, respectively. The ages of nTg mice are as described in Fig. 2. Figure S4. PAD2 expression is increased progressively in astrocytes in the spinal cord white matter of ALS mouse models. (A) Double immunofluorescence staining for GFAP and PAD2 in spinal cord white matter in SOD1G93A mice. (B, C) Quantification of fluorescent intensity of GFAP and PAD2, respectively, in (A). (D) Double immunofluorescence staining for GFAP and PAD2 in spinal cord white matter in PFN1C71G mice. (E, F) Quantification of fluorescent intensity of GFAP and PAD2, respectively, in (D). The ages of nTg mice, n, and statistics are as described in Fig. 2. Figure S5. PAD2 and citrullinated proteins are colocalized in astrocytes but not in aggregates in the spinal cord of ALS mouse models. (A, B) Double immunofluorescence staining for PAD2 and citrullinated proteins in the ventral horn gray matter of SOD1G93A and PFN1C71G mice, respectively. (C, D) Double immunofluorescence staining for PAD2 and citrullinated proteins in the ventral lateral white matter of SOD1G93A and PFN1C71G mice, respectively. Arrowheads point to PAD2-negative but citrulline-positive protein aggregates. The ages of nTg mice are as described in Fig. 2. Figure S6. PAD2 expression is not increased in microglia in the spinal cord of ALS mouse models. (A, B) Double immunofluorescence staining for IBA1 and PAD2 in ventral horn gray matter of SOD1G93A and PFN1C71G mice, respectively. (C, D) Double immunofluorescence staining for IBA1 and PAD2 in ventral lateral white matter of SOD1G93A and PFN1C71G mice, respectively. The ages of nTg mice are as described in Fig. 2. The blue color in the merge panel represents the nucleus as stained by DAPI. Figure S7. PAD2 expression is not increased in oligodendrocytes in the spinal cord of ALS mouse models. (A, B) Double immunofluorescence staining for Olig2 and PAD2 in ventral horn gray matter of SOD1G93A and PFN1C71G mice, respectively. (C, D) Double immunofluorescence staining for Olig2 and PAD2 in ventral lateral white matter of SOD1G93A and PFN1C71G mice, respectively. The ages of nTg mice are as described in Fig. 2. The blue color in the merge panel represents the nucleus as stained by DAPI. Figure S8. Disease-specific protein aggregates are observed in spinal cords from ALS mouse models. (A) Filter trap assay and quantification of mutant SOD1 protein aggregation. (B) Filter trap assay and quantification of mutant PFN1 protein aggregation. n = 4 in each group. Statistics: unpaired t-test for comparing transgenic mice with their age matched controls. *p < 0.05. ****p < 0.0001. Figure S9. Microscopy and Image processing workflow for quantitative analyses of immunofluorescence-stained spinal cord sections. (A) An example for quantifying PAD2 fluorescent intensity in astrocytes in ventral horn gray matter of a paralyzed PFN1C71G mouse. GFAP was used as a marker to identify astrocytes. The GFAP (red) image was thresholded using ImageJ, and GFAP-positive areas were identified as regions of interest (ROI). The ROI was overlayed on the corresponding immunostained PAD2 (green) image, and PAD2 fluorescent intensity was quantified exclusively within the defined ROI. (B) As in (A) but showing white matter. (C) An example of quantifying citrullinated protein fluorescent intensity in axons of nTg spinal cord white matter. NF-L was used as a marker to identify axons. The NF-L (Green) image was thresholded and NF-L-positive areas were identified as ROI. These ROIs were overlayed on the immunostained citrullinated proteins (red) image, and citrullinated protein fluorescent intensity was quantified exclusively within the defined ROI. (D) Same as (C) but in white matter of a paralyzed PFN1C71G mouse. Figure S10. Procedure for colocalization analysis in the spinal cord of ALS mouse models. Levels of colocalization between citrullinated protein aggregates and other proteins were analyzed using JACoP plugin in ImageJ. (A) PLP- and citrullinated protein-stained images (top) were thresholded to remove background and other weakly stained signals (bottom). (B) The Costes’ mask was generated showing colocalization (white), background (black), PLP (red), and citrullinated proteins (green). The result (colocalization) was expressed as Manders’ overlap coefficient (M2).

Additional file 1: Table S1. List of Primary Antibodies used in the Study. Table S2. List of Secondary Antibodies used in the Study.