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

Article Title: Citrullination of CXCL12 differentially reduces CXCR4 and CXCR7 binding with loss of inflammatory and anti-HIV-1 activity via CXCR4.

doi: 10.4049/jimmunol.182.1.666

Figure Lengend Snippet: FIGURE 1. Immunohistochemical detection of CXCL12 and PAD expression in the normal gastroin- testinal tract and in colon affected by Crohn’s disease. In the biopsy shown, the disease is severely active (score 10/16). A and B show immunohistochemical detection of CXCL12 expression in normal colon and colon tissue affected by Crohn’s disease, respectively. The epithelial staining for CXCL12 is most prominent in the mucosa adjacent to ulcerations and erosions and decreases to normal already at a short distance from the mucosal de- fect. C illustrates expression of PAD in a biopsy from a patient with Crohn’s disease. As observed for CXCL12, PAD expression is most distinct in the lesions. D shows a negative control staining of colon tissue affected by Crohn’s disease in which the primary Ab against PAD was omitted. Immunoreactive sites are stained bright red in A–D and these microphotographs were taken with a 5 objective. E and F show coexpression of CXCL12 and PAD in autoimmune disease (20 and 40 objec- tive). The colon biopsy of a patient with Crohn’s disease was stained with Abs against CXCL12 (red) and PAD (brown).

Article Snippet: Staining with Abs directed against CXCL12 (mouse monoclonal antihuman CXCL12, clone 79018; R&D Systems) and against human PAD (rabbit polyclonal anti-PAD, catalog no. NB100-1853; Novus Biologicals) was performed on formalin-fixed, paraffin-embedded tissue samples from the normal small intestine and colon, obtained from patients operated for diverticular disease and on biopsies from the small intestine and colon obtained from patients operated for Crohn’s disease.

Techniques: Immunohistochemical staining, Expressing, Staining, Negative Control

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

Article Title: Citrullination of CXCL12 differentially reduces CXCR4 and CXCR7 binding with loss of inflammatory and anti-HIV-1 activity via CXCR4.

doi: 10.4049/jimmunol.182.1.666

Figure Lengend Snippet: FIGURE 2. Conversion of CXCL12 by PAD. CXCL12 was treated with PAD at an enzyme:substrate ratio of 1:20 for 5–45 min at 37°C and the percent conversion of arginine to Cit on positions 8 (), 12 (), and 20 (‚) was determined by Edman degradation.

Article Snippet: Staining with Abs directed against CXCL12 (mouse monoclonal antihuman CXCL12, clone 79018; R&D Systems) and against human PAD (rabbit polyclonal anti-PAD, catalog no. NB100-1853; Novus Biologicals) was performed on formalin-fixed, paraffin-embedded tissue samples from the normal small intestine and colon, obtained from patients operated for diverticular disease and on biopsies from the small intestine and colon obtained from patients operated for Crohn’s disease.

Techniques:

Journal: Cells

Article Title: Poly(ADP-Ribose) Polymerase-1 Lacking Enzymatic Activity Is Not Compatible with Mouse Development

doi: 10.3390/cells12162078

Figure Lengend Snippet: The catalytic activity of PARP1-ΔC cells. ( A ) Immunofluorescent staining of PAR in MEFs of the indicated genotypes without (sham) or with the H 2 O 2 treatment (1 mM, 15 min) in the presence or absence of PARP inhibitor Olaparib (10 μM, 2 h). Scale bar: 20 μm. n = 8 . ( B ) Quantification of PAR intensity, n = 4. Statistics by ordinary one-way ANOVA were used; ** p < 0.01, **** p < 0.0001, ns, not significant. ( C , D ) Western blot analysis of MEF cells of the indicated genotypes without (sham) or with the H 2 O 2 treatment (1 mM, 15 min) in the presence or absence of Olaparib (10 μM, 15 min). The antibodies used are indicated. γ-tubulin is a loading control. n = 4. ( E ) Quantification of PARP2 protein levels in MEFs, normalised to γ-tubulin. The fold change is normalised to PARP2 levels in PARP1 +/ind− genotype. n = 4. Statistics by ordinary one-way ANOVA were used; * p < 0.05. ( F ) Quantification of NAD + levels ES cells of the indicated genotype without (sham) or with the H 2 O 2 treatment of (1 mM, 15 min). n = 4. Statistics by ordinary one-way ANOVA were used; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, not significant.

Article Snippet: The following antibodies and dilutions were used for immunoblotting: PAR 1:1000 (#4336-BPC-100, Trevigen), GAPDH 1:5000 (#G8795, Sigma-Aldrich), PARP1 C-terminus 1:1000 (C2-10, #4338-MC-50, Trevigen), PARP1 N-terminus 1:1000 (#9542, Cell Signalling, Danvers, MA, USA), PARP2 1:1200 (#55149-1-AP, Proteintech, Rosemont, IL, USA), γ-tubulin 1:5000 (#T6557, Sigma-Aldrich), p53 1:1000 (#2524, Cell Signalling), LC3B 1:2000 (#L7543, Sigma-Aldrich), GFP 1:2000 (#2956, Cell Signalling), KPL HRP-goat-anti-mouse 1:5000 (#5220-0341, SeraCare, Milford, MA, USA), and KPL HRP-goat-anti-rabbit 1:5000 (#5220-0336, SeraCare).

Techniques: Activity Assay, Staining, Western Blot, Control

Journal: Cells

Article Title: Poly(ADP-Ribose) Polymerase-1 Lacking Enzymatic Activity Is Not Compatible with Mouse Development

doi: 10.3390/cells12162078

Figure Lengend Snippet: Trapping of PARP1-ΔC and PARP2 proteins under genotoxic stress in ES cells. ( A ) Western blot analysis of a chromatin-bound fraction of PARP1-FL, PARP1-ΔC, and PARP2 proteins with MNNG (100 μM, 15 min) and/or PARP inhibitor Talazoparib (10 μM, 15 min). H3 is a loading control. n = 3. The red star indicates a non-specific band. ( B , C ) Quantification of PARP1-FL, PARP1-ΔC, and PARP2 protein levels after MNNG treatment and/or Talazoparib. The protein levels were normalised to H3 and then to sham controls. Statistics by ordinary one-way ANOVA were used; * p < 0.05, ** p < 0.01, **** p < 0.0001, ns, not significant. ( D ) The proliferation of ES cell lines in the presence or absence of Olaparib (PARPi, 2 μM). n = 5. Statistics by one-way repeated measures nonparametric test, matched rows with Friedman test were used to analyse the mean values within individual days, * p < 0.05, **** p < 0.0001.

Article Snippet: The following antibodies and dilutions were used for immunoblotting: PAR 1:1000 (#4336-BPC-100, Trevigen), GAPDH 1:5000 (#G8795, Sigma-Aldrich), PARP1 C-terminus 1:1000 (C2-10, #4338-MC-50, Trevigen), PARP1 N-terminus 1:1000 (#9542, Cell Signalling, Danvers, MA, USA), PARP2 1:1200 (#55149-1-AP, Proteintech, Rosemont, IL, USA), γ-tubulin 1:5000 (#T6557, Sigma-Aldrich), p53 1:1000 (#2524, Cell Signalling), LC3B 1:2000 (#L7543, Sigma-Aldrich), GFP 1:2000 (#2956, Cell Signalling), KPL HRP-goat-anti-mouse 1:5000 (#5220-0341, SeraCare, Milford, MA, USA), and KPL HRP-goat-anti-rabbit 1:5000 (#5220-0336, SeraCare).

Techniques: Western Blot, Control