Fig 1: Haematopoiesis-specific deletion of PADI4 has no impact on BM reconstitution potential following serial transplantations. (A) Experimental design for BM transplantation experiments. 200 CD45.2+ BM HSCs from C57BL/6 Padi4CTL or Padi4CKO mice were transplanted into primary recipient mice and monitored for 16 weeks. Following this, a cohort of mice were sacrificed for analysis at 16 weeks post-transplantation and bone marrow was transplanted to secondary recipients. (B) Percentage of donor-derived CD45.2+ cells in total BM, LSK, LK, HSC, MPP, HPC-1, HPC-2 and differentiated cell populations (granulocytes, monocytes, erythroid and B cells). Padi4CTL, n=14; Padi4CKO, n=13. (C) Contribution of donor-derived CD45.2+ cell population to total spleen WBC count and differentiated cell populations of primary recipients. Padi4CTL, n=6; Padi4CKO, n=6. (D,E) Secondary recipient mice were transplanted with 3000 sorted CD45.2+ BM LSK cells from primary recipients euthanised at 16 weeks. (D) Percentage of donor-derived CD45.2+ cells in total BM, LSK, LK, HSC, MPP, HPC-1, HPC-1 and differentiated cell lineages (granulocytes, monocytes, erythroid and B cells). Padi4CTL, n=19; Padi4CKO, n=19. Two to four donors were used per genotype. (E) Contribution of donor-derived CD45.2+ cell population to spleen WBC and differentiated cells of secondary recipients. Padi4CTL, n=19; Padi4CKO, n=19. All data are mean±s.e.m. *P<0.05; **P<0.01; ***P<0.001, ****P<0.0001 (Mann–Whitney U-test).
Fig 2: The interaction between citrullinated histone and monoclonal ACPA induce innate responses. The immune stimulatory effect of seven ACPA monoclonal with high cit-histone 2B binding (1325:01B09, 37CEPT1G09, 37CEPT2C04), medium binding (1325:04C03, 1325:05C06, 14CFCT3G09), and no or low binding (14CFCT2D09) was evaluated using plate-bound immune complexes at 10 µg/ml with PAD4 citrullinated histone 2B. Healthy donor PBMC were incubated with the antigen-captured IgG plates for 20 h at 37°C and IL-8 (A) and TNF-a (C) expression was subsequently assessed in the cell supernatants by ELISA. The importance of Fc?R interaction for 1325:01B09-mediated cytokine stimulations was assessed by comparison to the Fc?R-null binding genetic variant 1325:01B09 GRLR (B,D). Both recombinant RA derived human IgG (1276:01G09, Control IgG-b) and commercial isotype control IgG (ET901, Biolegend, Control IgG-a) were used as controls. The figure show values in triplicate for one representative donor. Data for multiple donors are available in Supplementary Figure 5. P-values are derived from student t-test.
Fig 3: C3a is an important molecule in IRI. (A) Renal levels of C3a from WT and C3KO mice, measured by ELISA. (B) Immunoblotting analysis of C3aR expression in mouse kidneys 24 h after IRI. (C) Quantitative analysis was analyzed by normalization to β-actin expression, n = 3/group. (D) Immunoblotting analysis of PAD4 expression in mouse kidneys 24 h after IRI. (E) Quantitative analysis was analyzed by normalization to β-actin expression, n = 3/group. *P < 0.05, **P < 0.01. Data are presented as mean ± SD.
Fig 4: (A) miR-155-5p expression and (B) PAD4 mRNA expression. Neutrophils were stimulated with or without PMA (500 nM). Relative expression was determined by qRT-PCR. U6 and GAPDH were used as a house-keeping genes to normalize miR-155-5p and PAD4 expression, respectively. Relative expression was determined using 2−ΔΔCT method. Data represent mean ± SEM and n = 5. *P < 0.05 vs. control.
Fig 5: Levels of DNA histone complexes in neutrophils transfected with (A) Ctrl Mimic or mir-155 mimic and (B) Ctrl-antagomiR or antagomiR-155-5p. Expression of (C) PAD4 and (D) citH3 protein as determined by western blot and aggregate data showing PAD4 and citH3 protein expression normalized to the total protein of the respective lane. Band intensity was quantified by using Image Lab™ software. Western blots are representative of 4 independent experiments. Data are expressed as mean ± SEM and n = 4–5 and represented as fold change. #P < 0.05 vs. control and *P < 0.05 vs. vehicle.
Supplier Page from Abcam for Anti-PADI4 / PAD4 antibody [EPR20706]