Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Citrullination alters immunomodulatory function of LL-37 essential for prevention of endotoxin-induced sepsis

doi: 10.4049/jimmunol.1303062

Figure Lengend Snippet: Human MDMs (A) and mouse RAW 264.7 (B) macrophages were stimulated with 10 ng/mL LPS in the presence of native or citrullinated LL-37 at the indicated concentrations (0.1–10 μg/mL). Citrullinated LL-37 was obtained by treatment of the native peptide with human PAD2 or PAD4 at 23.3 U/mg peptide). The level of TNF-α (A) and NO (B) in the culture supernatants was determined using ELISA or the Griess assay at 6 or 20 h post-stimulation, respectively. Since neither the LL-37 nor the PAD enzymes alone induced the release of NO or TNF-α, for the sake of clarity these controls are not shown in the figure. Data represent the mean ± SD of three independent experiments. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: Recombinant human PAD2 and PAD4 were obtained from Modiquest (The Netherlands).

Techniques: Enzyme-linked Immunosorbent Assay, Griess Assay

Journal: bioRxiv

Article Title: Citrullination of TDP-43 is a key post-translation modification associated with structural and functional changes and progressive pathology in TDP-43 mouse models and human proteinopathies

doi: 10.1101/2025.02.28.639952

Figure Lengend Snippet: a, The enzymatic reaction mediated by PADs converts arginine to citrulline. b, c, Western blot images of PAD2 and PAD4-mediated citrullination of H3 (b) and TDP-43 (c). In (c) arrows indicate the observed shift in TDP-43 molecular weight. d, Coomassie staining of PAD2 (bands 1, 4), PAD4 (bands 2, 6), unmodified TDP-43 (band 3), PAD2-mediated citR TDP-43 (band 5), and PAD4-mediated citR TDP-43 (band 7). e, Bar diagrams showing the MW shift (kDa) of TDP-43 protein following citrullination. f, Schematic representation of arginine epitopes positioned in the human TDP-43 protein sequence. Position of the 11 citrullinated arginine epitopes are indicated in red. g, Six out of eleven arginine epitopes susceptible to citrullination laid within common RX(X)R (red box) or RXG/RGGG (green box) motifs in TDP-43 sequence. h, MS/MS spectrum showing b- and y-ion coverage of modified citR83 peptide and extracted ion chromatograms (XICs) showed abundance and retention time of unmodified R83 vs. citR83 with PAD2 and PAD4 treatment (intact peptide monoisotopic m/z (+2) 719.8457, (+3) 480.2329). i, Retention time peaks for TDP-43 peptides surrounding the unmodified or modified R83 epitope and base peak m/z undergoing methionine (Met85) oxidation, citrullination or both showed reliable time separation.

Article Snippet: PAD2 and PAD4 primary antibodies were purchased from Proteintech, US (#12110-1-AP and 17373-1-AP) and diluted at 1:10000 vs. 1:1000, respectively.

Techniques: Western Blot, Molecular Weight, Staining, Sequencing, Tandem Mass Spectroscopy, Modification

Journal: bioRxiv

Article Title: Citrullination of TDP-43 is a key post-translation modification associated with structural and functional changes and progressive pathology in TDP-43 mouse models and human proteinopathies

doi: 10.1101/2025.02.28.639952

Figure Lengend Snippet: Spectra showing b- and y-ion coverage of citrullinated peptides, as well as XICs retention times for unmodified and citrullinated peptides treated with PAD2 and PAD4, corresponding to a - c, citR165 (intact peptide monoisotopic m/z (+2) 665.8214), d - f, citR191(intact peptide monoisotopic m/z (+2) 672.8469), g - i, citR268/272 (intact peptide monoisotopic m/z (+2) 728.3560), j - l, citR293 (intact peptide monoisotopic m/z (+2) 638.29).

Article Snippet: PAD2 and PAD4 primary antibodies were purchased from Proteintech, US (#12110-1-AP and 17373-1-AP) and diluted at 1:10000 vs. 1:1000, respectively.

Techniques:

Journal: bioRxiv

Article Title: Citrullination of TDP-43 is a key post-translation modification associated with structural and functional changes and progressive pathology in TDP-43 mouse models and human proteinopathies

doi: 10.1101/2025.02.28.639952

Figure Lengend Snippet: a, Immunohistochemical images of Non-Tg, TAR4 and TAR4/4 cortex labeled with PAD2 and PAD4 antibodies. b, Fold change of PAD2 and PAD4 (% area mean of the values ± SEM) normalized to the Non-Tg control. One-way or Two-way ANOVA, followed by Tukey’s post hoc multiple comparisons tests, n = 5, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. c, Schematic overview of the TAR TDP-43 mouse model and the fractionation of cortical tissue into RIPA-soluble and (7 M) Urea-soluble fractions. d, The expression levels of the human TDP-43 in the TAR model. e, Cortical RIPA soluble and f, Urea soluble fraction analyzed by Western blotting and probed for citR TDP-43 antibody panel (citR83, 165, 191, 268/272, 275), pTDP-43 409/410 and total human TDP-43 protein. g, Quantification of citR TDP-43 43 kDa protein levels, proteolytic fragments (17, 25 & 35 kDa) and intermediate high molecular species (72, 98 & 120k Da) normalized to GAPDH in RIPA and h, Urea fraction normalized to the total protein loaded signal. Data represent the mean of the values ± SEM; One-way ANOVA, followed by Tukey’s or Šidák post hoc multiple comparisons tests, n = 5, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: PAD2 and PAD4 primary antibodies were purchased from Proteintech, US (#12110-1-AP and 17373-1-AP) and diluted at 1:10000 vs. 1:1000, respectively.

Techniques: Immunohistochemical staining, Labeling, Control, Fractionation, Expressing, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Peptidyl arginine deiminase immunization induces anticitrullinated protein antibodies in mice with particular MHC types

doi: 10.1073/pnas.1713112114

Figure Lengend Snippet: IgG responses to PADs in C3H mice. IgG responses to murine PAD2 or human PAD2 or human PAD4 were analyzed by ELISAs. Plates were coated with PADs and blocked with BSA. Sera from primed mice were obtained at 15, 35, 55, and 65 d postimmunization and were diluted at 1/40. ( A ) For PBS-immunized mice, 15 sera were tested for murine PAD2, 8 sera were tested for human PAD2, and 9 sera were tested for human PAD4. ( B ) For PAD-immunized mice, each serum was tested against the same PAD used for each immunization. After washing, peroxidase-conjugated antimurine IgG was added. The OD was read at 405 nm. The background OD was obtained by adding each serum to a well without PAD (negative). Positive sera were defined as an OD value higher than twice the background OD.

Article Snippet: Murine PAD2 and PAD4 and human PAD2 and PAD4 proteins were purchased from Proteogenix.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Peptidyl arginine deiminase immunization induces anticitrullinated protein antibodies in mice with particular MHC types

doi: 10.1073/pnas.1713112114

Figure Lengend Snippet: IgG responses to PADs in DBA/2 mice. Plates were coated with PADs and blocked with BSA. Sera from primed mice were obtained at 15, 35, 55, and 65 d postimmunization and were diluted at 1/40. ( A ) For PBS-immunized mice, nine sera were tested for murine PAD2 and human PAD4. ( B ) For PAD-immunized mice, each serum was tested against the same PAD used for each immunization. After washing, peroxidase-conjugated antimurine IgG was added. The OD was read at 405 nm. The background OD was obtained by adding each serum to a well without PAD (negative). Positive sera were defined as an OD value higher than twice the background OD.

Article Snippet: Murine PAD2 and PAD4 and human PAD2 and PAD4 proteins were purchased from Proteogenix.

Techniques:

Journal: Biology

Article Title: Extracellular Vesicles and Post-Translational Protein Deimination Signatures in Mollusca—The Blue Mussel ( Mytilus edulis ), Soft Shell Clam ( Mya arenaria ), Eastern Oyster ( Crassostrea virginica ) and Atlantic Jacknife Clam ( Ensis leei )

doi: 10.3390/biology9120416

Figure Lengend Snippet: Deiminated proteins in hemolymph of blue mussel ( Mytilus edulis ), as identified by F95-enrichment in conjunction with LC–MS/MS analysis. Deiminated proteins were isolated from hemolymph (a pool of hemolymph from four individual animals) by immunoprecipitation using the pan-deimination F95 antibody. The resulting F95-enriched eluate was then analyzed by LC–MS/MS and peak list files submitted to Mascot, using both a species-specific as well as a common Mollusca database. Peptide sequence hits are listed, showing species-specific hits, number of sequences for protein hits, and total score. Species hit names are indicated, blue mussel specific hits are on the top of the list and highlighted. *Proteins only identified in blue mussel. (See Table S1 for full details on all protein hits).

Article Snippet: Primary antibody incubation was carried out overnight at 4 °C on a shaking platform using the following antibodies for Mollusca sera: F95 pan-deimination antibody (MABN328, Merck; diluted 1/1000 in TBS-T) and anti-human PAD2 antibody (anti-PAD2, ab50257, Abcam, Cambridge, UK; diluted 1/1000), for detection of putative PAD protein homologues, due to PAD2 being the most conserved PAD isozyme and the anti-human PAD2 antibody was previously shown to cross-react with PADs across taxa [ , , , , , , , , , , , , , ].

Techniques: Isolation, Immunoprecipitation, Sequencing, Binding Assay

Journal: Biology

Article Title: Extracellular Vesicles and Post-Translational Protein Deimination Signatures in Mollusca—The Blue Mussel ( Mytilus edulis ), Soft Shell Clam ( Mya arenaria ), Eastern Oyster ( Crassostrea virginica ) and Atlantic Jacknife Clam ( Ensis leei )

doi: 10.3390/biology9120416

Figure Lengend Snippet: Deiminated proteins in hemolymph of soft shell clam ( Mya arenaria ), as identified by F95-enrichment in conjunction with LC–MS/MS analysis. Deiminated proteins were isolated from hemolymph (a pool of hemolymph from four individual animals) by immunoprecipitation using the pan-deimination F95 antibody. The resulting F95-enriched eluate was then analyzed by LC–MS/MS and peak list files submitted to Mascot, using both a species-specific as well as a common Mollusca database. Peptide sequence hits are listed, showing species-specific hit, number of sequences for protein hits, and total score. Species hit names are indicated, soft shell clam specific hits are on the top of the list and highlighted. * Proteins only identified in soft shell clam. (See Table S2 for full details on all protein hits).

Article Snippet: Primary antibody incubation was carried out overnight at 4 °C on a shaking platform using the following antibodies for Mollusca sera: F95 pan-deimination antibody (MABN328, Merck; diluted 1/1000 in TBS-T) and anti-human PAD2 antibody (anti-PAD2, ab50257, Abcam, Cambridge, UK; diluted 1/1000), for detection of putative PAD protein homologues, due to PAD2 being the most conserved PAD isozyme and the anti-human PAD2 antibody was previously shown to cross-react with PADs across taxa [ , , , , , , , , , , , , , ].

Techniques: Isolation, Immunoprecipitation, Sequencing

Journal: Biology

Article Title: Extracellular Vesicles and Post-Translational Protein Deimination Signatures in Mollusca—The Blue Mussel ( Mytilus edulis ), Soft Shell Clam ( Mya arenaria ), Eastern Oyster ( Crassostrea virginica ) and Atlantic Jacknife Clam ( Ensis leei )

doi: 10.3390/biology9120416

Figure Lengend Snippet: Deiminated proteins in hemolymph of Eastern oyster ( Crassostrea virginica ), as identified by F95-enrichment followed by LC–MS/MS analysis. Deiminated proteins were isolated from hemolymph (a pool of hemolymph from four individual animals) by immunoprecipitation using the pan-deimination F95 antibody. The resulting F95-enriched eluate was then analyzed by LC–MS/MS and peak list files submitted to Mascot, using both a species-specific as well as a common Mollusca database. Peptide sequence hits are listed, showing species-specific hit, number of sequences for protein hits, and total score. Species hit names are indicated, Eastern oyster specific hits are on the top of the list and highlighted. * Proteins only identified in Eastern oyster. (See Table S3 for full details on all protein hits).

Article Snippet: Primary antibody incubation was carried out overnight at 4 °C on a shaking platform using the following antibodies for Mollusca sera: F95 pan-deimination antibody (MABN328, Merck; diluted 1/1000 in TBS-T) and anti-human PAD2 antibody (anti-PAD2, ab50257, Abcam, Cambridge, UK; diluted 1/1000), for detection of putative PAD protein homologues, due to PAD2 being the most conserved PAD isozyme and the anti-human PAD2 antibody was previously shown to cross-react with PADs across taxa [ , , , , , , , , , , , , , ].

Techniques: Isolation, Immunoprecipitation, Sequencing

Journal: Biology

Article Title: Extracellular Vesicles and Post-Translational Protein Deimination Signatures in Mollusca—The Blue Mussel ( Mytilus edulis ), Soft Shell Clam ( Mya arenaria ), Eastern Oyster ( Crassostrea virginica ) and Atlantic Jacknife Clam ( Ensis leei )

doi: 10.3390/biology9120416

Figure Lengend Snippet: Deiminated proteins in hemolymph of Atlantic jacknife clam ( Ensis leei ), as identified by F95-enrichment followed by LC–MS/MS analysis. Deiminated proteins were isolated from hemolymph (a pool of hemolymph from four individual animals) by immunoprecipitation using the pan-deimination F95 antibody. The resulting F95-enriched eluate was then analyzed by LC–MS/MS and peak list files submitted to Mascot, using both a species-specific as well as a common Mollusca database. Peptide sequence hits are listed, showing species-specific hit, number of sequences for protein hits, and total score. * Proteins only identified in Atlantic jacknife clam. (See Table S4 for full details on all protein hits).

Article Snippet: Primary antibody incubation was carried out overnight at 4 °C on a shaking platform using the following antibodies for Mollusca sera: F95 pan-deimination antibody (MABN328, Merck; diluted 1/1000 in TBS-T) and anti-human PAD2 antibody (anti-PAD2, ab50257, Abcam, Cambridge, UK; diluted 1/1000), for detection of putative PAD protein homologues, due to PAD2 being the most conserved PAD isozyme and the anti-human PAD2 antibody was previously shown to cross-react with PADs across taxa [ , , , , , , , , , , , , , ].

Techniques: Isolation, Immunoprecipitation, Sequencing