Journal: Journal of thrombosis and haemostasis : JTH

Article Title: The lectin-like domain of thrombomodulin interferes with complement activation and protects against arthritis.

doi: 10.1111/j.1538-7836.2006.02033.x

Figure Lengend Snippet: Fig. 4. Complement C3 deposition in joints. Healthy 5–8-week old female TMwt/wt (A) and TMLeD/LeD (B) mice were killed and knee joints were processed for immunohistochemical staining to detect complement factor C3 deposition. These sections are representative of three mice. No C3 was detectable in the joints of TMwt/wt mice (A). C3 was readily observed on the articular surface (small arrows) and in the bone marrow space of TMLeD/LeD mice (large arrows) (B). Higher power views of the articular surface are shown in the right upper corner of each panel.

Article Snippet: Specific immunostaining of histologic sections was achieved by overnight incubation with the following primary antibodies: CD45 (rat, 1:100; BecktonDickinson, Erembodegem, Belgium, no. 553076);Mac3 (rat, 1:300; BecktonDickinson, no. 553322); MPO (rabbit, 1:100, Dako A/S, Glostrup, Denmark, no. A0398); HMGB1 (rabbit; 1:250, Santa Cruz Biotechnology, Boechout, Belgium, no. sc12523); and complement factor C3 (rat, 1:200; Cedarlane,Uden, theNetherlands, no. CL7503AP), which recognizes intact C3, C3b, iC3b and C3d, but not C3a.

Techniques: Immunohistochemical staining, Staining

Journal: Frontiers in Immunology

Article Title: Complement Plays a Critical Role in Inflammation-Induced Immunoprophylaxis Failure in Mice

doi: 10.3389/fimmu.2021.704072

Figure Lengend Snippet: Complement is fixed on KEL RBCs in-vivo and in-vitro in response to polyclonal anti-KEL (KELIg). (A) General in-vivo experimental design: recipients were treated with or without KELIg and transfused with DiO labeled KEL RBCs. (B) C3, C4, and Factor B were measured on recovered DiO positive KEL RBCs 1-hour post-transfusion; black filled histogram shows condition without KELIg; grey shaded histogram shows condition with KELIg. (C) In-vitro experiments were completed with KELIg incubated with KEL RBCs in the presence (darkest histogram) or absence (lightest histogram) of serum; an additional condition included saline incubated with KEL RBCs in the presence of serum (medium grey histogram). (D) Additional in-vitro conditions included KELIg incubated with KEL RBCs in the presence of serum (darkest histogram), in the presence of serum with EGTA-Mg (lightest histogram) or serum with EDTA (medium grey histogram). These data are representative of 3 independent experiments; p < 0.05 for C3 detection on RBCs in the presence or absence of KELIg.

Article Snippet: Following transfusion of KEL RBCs, complement was evaluated on the visualized RBC surface using antibodies against C3 (Cl7503B, Cedarlane, Ontario, Canada), C4 (clone 16D2, Santa Cruz), or Factor B (CL8824AP, Cedarlane, Ontario, Canada) followed by streptavidin or a fluorescently conjugated donkey anti-rabbit antibody (BioLegend, San Diego, California).

Techniques: In Vivo, In Vitro, Labeling, Incubation, Saline

Journal: Frontiers in Immunology

Article Title: Complement Plays a Critical Role in Inflammation-Induced Immunoprophylaxis Failure in Mice

doi: 10.3389/fimmu.2021.704072

Figure Lengend Snippet: Recipient C3 and C1q contribute to KELIg immunoprophylaxis failure in the setting of poly (I:C). (A) General in-vivo experimental design: recipients were infused with KELIg and transfused the following day in the absence or presence of poly (I:C) treatment, with anti-KEL responses evaluated longitudinally. (B) Anti-KEL IgG responses in wild type compared with C3 -/- recipients transfused in the absence of poly (I:C). (C) Anti-KEL IgG responses in wild type compared with C3 -/- recipients transfused in the presence of poly (I:C). (D) Anti-KEL IgG responses in wild type compared with C1q -/- recipients transfused in the presence of poly (I:C). (E) Baseline impact of recipient treatment with poly (I:C) on alloimmune responses to KEL RBCs in the absence of KELIg. These data are representative of 2-3 independent experiments, with 3-5 mice/group/experiment; error bars indicate standard deviation between individual mice. *p < 0.05 (C, D) d14,21,28, and all comparisons for (E) .

Article Snippet: Following transfusion of KEL RBCs, complement was evaluated on the visualized RBC surface using antibodies against C3 (Cl7503B, Cedarlane, Ontario, Canada), C4 (clone 16D2, Santa Cruz), or Factor B (CL8824AP, Cedarlane, Ontario, Canada) followed by streptavidin or a fluorescently conjugated donkey anti-rabbit antibody (BioLegend, San Diego, California).

Techniques: In Vivo, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Complement Plays a Critical Role in Inflammation-Induced Immunoprophylaxis Failure in Mice

doi: 10.3389/fimmu.2021.704072

Figure Lengend Snippet: Recipient complement, KEL RBC clearance, and KEL antigen expression. KEL RBCs were labeled with DiO and mixed with wild type RBCs labeled with DiI; this mixture was transfused into recipients that had been infused with KELIg and treated with poly (I:C). (A) shows wild type compared with C3 -/- recipients; (B) shows wild type compared with C1q -/- recipients. Recovered DiO labeled RBCs were then evaluated for KEL glycoprotein expression by flow cytometry, after incubation with KELIg and fluorescently conjugated anti-mouse IgG; (C) shows wild type recipients compared with C3 -/- recipients; (D) shows wild type recipients compared with C1q -/- recipients. These data are representative of 2-3 independent experiments with 3 mice/group/experiment; error bars indicate standard deviation between mice. *p < 0.05 for all comparisons in (C) and p = ns, not significant for all comparisons in (D) .

Article Snippet: Following transfusion of KEL RBCs, complement was evaluated on the visualized RBC surface using antibodies against C3 (Cl7503B, Cedarlane, Ontario, Canada), C4 (clone 16D2, Santa Cruz), or Factor B (CL8824AP, Cedarlane, Ontario, Canada) followed by streptavidin or a fluorescently conjugated donkey anti-rabbit antibody (BioLegend, San Diego, California).

Techniques: Expressing, Labeling, Flow Cytometry, Incubation, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Complement Plays a Critical Role in Inflammation-Induced Immunoprophylaxis Failure in Mice

doi: 10.3389/fimmu.2021.704072

Figure Lengend Snippet: Poly (I:C) and KELIg immunoprophylaxis result in increased transfused KEL RBC consumption by splenic inflammatory monocytes in wild type compared with C3 -/- mice. DiO labeled KEL RBCs were transfused to wild type or C3 -/- mice treated with KELIg in the presence or absence of poly (I:C) and splenic cell subsets were evaluated at 1 and 16 hours post-transfusion (A, B) . (C) Representative histograms for DiO RBC fluorescence patterns of inflammatory monocytes at 16 hours post-transfusion, after first excluding TER119 positive RBCs on the exterior of the splenic cells; black open histogram is KELIg in wild type, dotted open histogram is KELIg and PIC in wild type; white shaded histogram is KELIg in C3 -/- and dotted shaded histogram is KELIg and PIC in C3 -/- . DiO mean fluorescence intensity (MFI) of the splenic cell subsets was evaluated at 1 hour (D) and 16 hours (E) post-transfusion. These data are representative of 2-3 independent experiments with 3 mice/group/experiment; error bars indicate standard deviation between mice.

Article Snippet: Following transfusion of KEL RBCs, complement was evaluated on the visualized RBC surface using antibodies against C3 (Cl7503B, Cedarlane, Ontario, Canada), C4 (clone 16D2, Santa Cruz), or Factor B (CL8824AP, Cedarlane, Ontario, Canada) followed by streptavidin or a fluorescently conjugated donkey anti-rabbit antibody (BioLegend, San Diego, California).

Techniques: Labeling, Fluorescence, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Complement Plays a Critical Role in Inflammation-Induced Immunoprophylaxis Failure in Mice

doi: 10.3389/fimmu.2021.704072

Figure Lengend Snippet: Complement-fixed KEL RBCs bind in-vitro to wild type donor derived B-cells but not to CR1/2 -/- B-cells. DiO labeled KEL RBCs were incubated with KELIg in the presence of sera, followed by incubation with peripheral blood derived WBCs from donor mice. (A) In wells using WBCs from wild type donor mice, CD19+B220+ B-cells cells were separated by DiO positivity and then by C3 positivity. The CD19+B220+ cells were next separated by their CD23 and CD21/35 expression, with the cells highest for CD23 and CD21/35 gated in the larger gate and those less strongly positive for CD23 and CD21/35 gated in the smaller gate; the DiO positive and negative populations were then evaluated for their C3 positivity. Shaded histograms are the DiO positive population, open histograms are the DiO negative population. (B) In wells using WBCs from CR1/2 -/- mice, CD19+B220+ B-cells cells were separated by DiO positivity and then by C3 positivity. Shaded histograms are the DiO positive population, open histograms are the DiO negative population. These data are representative of more than 3 independent experiments, with 2 involving CD23 and CD21/35 staining.

Article Snippet: Following transfusion of KEL RBCs, complement was evaluated on the visualized RBC surface using antibodies against C3 (Cl7503B, Cedarlane, Ontario, Canada), C4 (clone 16D2, Santa Cruz), or Factor B (CL8824AP, Cedarlane, Ontario, Canada) followed by streptavidin or a fluorescently conjugated donkey anti-rabbit antibody (BioLegend, San Diego, California).

Techniques: In Vitro, Derivative Assay, Labeling, Incubation, Expressing, Staining

Journal: Frontiers in Immunology

Article Title: Complement Plays a Critical Role in Inflammation-Induced Immunoprophylaxis Failure in Mice

doi: 10.3389/fimmu.2021.704072

Figure Lengend Snippet: CR1/2 are required for KELIg immunoprophylaxis failure in the setting of poly (I:C). (A) KEL RBCs were transfused into wild type recipients or recipients lacking CR1/2, in the presence or absence of poly (I:C). (B) RBCs were labeled with DiO and mixed with wild type RBCs labeled with DiI; this mixture was transfused into wild type or CR1/2 -/- recipients that had been infused with KELIg and treated with poly (I:C). Recovered DiO positive KEL RBCs were evaluated for (C) KEL glycoprotein expression, and (D) bound complement C3. (E) Wild type or CR1/2 -/- recipients were infused with KELIg and transfused the following day in the absence or presence of poly (I:C), with anti-KEL responses evaluated longitudinally. *p < 0.05 for d14, 21 and 28 of (A) between CR1/2 -/- mice treated with or without poly (I:C); p < 0.05 for 10 min and 1-hour timepoints in (C) ; p < 0.05 for d14, 21, and 28 in (E) . These data are representative of 2-3 independent experiments with 3 mice/group/experiment; error bars indicate standard deviation between mice. ns, not significant.

Article Snippet: Following transfusion of KEL RBCs, complement was evaluated on the visualized RBC surface using antibodies against C3 (Cl7503B, Cedarlane, Ontario, Canada), C4 (clone 16D2, Santa Cruz), or Factor B (CL8824AP, Cedarlane, Ontario, Canada) followed by streptavidin or a fluorescently conjugated donkey anti-rabbit antibody (BioLegend, San Diego, California).

Techniques: Labeling, Expressing, Standard Deviation

Journal: Nature neuroscience

Article Title: The endogenous neuronal complement inhibitor SRPX2 protects against complement-mediated synapse elimination during development

doi: 10.1038/s41593-020-0672-0

Figure Lengend Snippet: a , Immunoprecipitation (IP) of SRPX2-FLAG from mouse brain lysate co-immunoprecipitated C1q but not C3, which indicates that SRPX2 and C1q are present in the same protein complex in vivo in WT and SRPX2-FLAG knock-in (KI) mice. This experiment was repeated independently 4 times. b , HEK293 cells were cotransfected with SRPX2-FLAG and HA-C1q proteins, lysed, immunoprecipitated with an anti-FLAG antibody and blotted for HA and FLAG. Anti-FLAG beads immunoprecipitated murine C1qa/b/c only in the presence of SRPX2-FLAG. This experiment was repeated independently 4 times. c , HEK293T cells were co-transfected with SRPX2-FLAG and HA-C1q, and cell medium was collected. After clearing, the medium was immunoprecipitated with anti-FLAG antibody, and blotted for HA and FLAG. HA-C1q was not co-immunoprecipitated with SRPX2-FLAG from the cell medium. This experiment was independently repeated 3 times. d , Increasing amounts of purified C1q, C3b and BSA were immobilized onto a nitrocellulose membrane, which was then incubated with conditioned medium containing SRPX2-FLAG, and then blotted with anti-FLAG. SRPX2-FLAG shows dose-dependent saturable binding to purified human C1q, but not to C3b or BSA. This experiment was repeated independently 3 times.

Article Snippet: Protein complexes were eluted with 100 μL of 3X FLAG peptide (200 ng/μL) according to the manufacturer’s protocol (Sigma-Aldrich), and the eluates were blotted with anti-FLAG M2-peroxidase (HRP) (Sigma-Aldrich, Cat. no. A8592-0.2mg), anti-C1Q (Abcam, Cat no. ab182451), and anti-C3 (MP Biomedicals, Cat. no. 0855730) antibodies.

Techniques: Immunoprecipitation, In Vivo, Knock-In, Transfection, Purification, Incubation, Binding Assay

Journal: Nature neuroscience

Article Title: The endogenous neuronal complement inhibitor SRPX2 protects against complement-mediated synapse elimination during development

doi: 10.1038/s41593-020-0672-0

Figure Lengend Snippet: a, RNAscope ISH for SRPX2 mRNA (red) in P10 dLGN and P60 L4 SS cortex, combined with immunostaining for cell-specific markers (green) for neurons (NeuN), microglia (Iba1), astrocytes (GFAP), and oligodendrocytes (Olig2). Nuclei were stained with DAPI (blue). Scale bars 20 μm. These experiments were repeated independently 4 times. b, Quantification of ISH for SRPX2 mRNA in P10 dLGN and P60 L4 SS cortex. The number of SRPX2 mRNA puncta colocalized with the DAPI area of each cell type was plotted as mean ± s.e.m.. Data were analyzed using one-way ANOVA with two-sided Dunnett’s post-hoc test comparing all means to NeuN. P10 dLGN: NeuN, n = 14, Iba1, n = 13 **** P < 0.0001, GFAP, n = 13 images **** P < 0.0001, Olig2, n = 14 **** P < 0.0001, all images from 3 mice each. P60 L4 of the SS cortex: NeuN, n = 15 images, Iba1, n = 16 images **** P < 0.0001, GFAP, n = 11 **** P < 0.0001, Olig2, n = 15 **** P < 0.0001;, all images from 3 mice each. c , Representative images of P10 LGN and P60 L4 SS cortex stained for SRPX2 (magenta) and complement components C1q and C3 (green), as well as synaptic markers VGlut1, VGlut2, and VGAT (green). Arrows indicate colocalized puncta of SRPX2 and C1q/VGlut1/VGlut2. Insets show magnified examples of colocalized puncta. Scale bars 10 μm. These experiments were repeated independently 3 times. d, Colocalization analysis of SRPX2 and various markers in P10 LGN and P60 L4 SS cortex. A Pearson’s coefficient cross-correlation function (CCF) was calculated with the green channel shifted pixel by pixel with respect to the red channel. SRPX2 is most colocalized with C1q.

Article Snippet: Protein complexes were eluted with 100 μL of 3X FLAG peptide (200 ng/μL) according to the manufacturer’s protocol (Sigma-Aldrich), and the eluates were blotted with anti-FLAG M2-peroxidase (HRP) (Sigma-Aldrich, Cat. no. A8592-0.2mg), anti-C1Q (Abcam, Cat no. ab182451), and anti-C3 (MP Biomedicals, Cat. no. 0855730) antibodies.

Techniques: Immunostaining, Staining

Journal: Nature neuroscience

Article Title: The endogenous neuronal complement inhibitor SRPX2 protects against complement-mediated synapse elimination during development

doi: 10.1038/s41593-020-0672-0

Figure Lengend Snippet: a , SRPX2 inhibition of complement activity was tested in the total complement activity (CH50) assay. SRPX2-FLAG immune-isolated from transfected HEK cells inhibited the generation of TCCs in normal human serum after activation of the classical complement pathway, as assessed using the CH50 enzyme-linked immunoassay (Quidel). Data are shown as mean ± s.e.m., and analyzed by two-tailed unpaired t -test, **** P <0.0001, n = 5. b , SRPX2-FLAG immune-isolated from transfected HEK293T cells was incubated with C1 and C2, and the cleavage of C2 was assessed by blotting for C2 fragments. As a negative control, C1 was omitted from the reaction. As a positive control for inhibition of C2 cleavage, a C1 inhibitor (C1-INH) was added. c , Intensities of bands representing cleaved C2 as a fraction of total C2 bands in the lane are plotted as means ± s.e.m., and analyzed by two-tailed unpaired t -test, *** P = 0.0005, n = 3. d,e , Representative images of P10 dLGN ( d ) and P60 L4 SS cortex ( e ) of SRPX2 +/Y and SRPX2 −/Y mice stained for C1q and C3. Scale bar 10 μm. These experiments were repeated independently 3-4 times. f,g , Quantitation of C1q and C3 levels in P10 dLGN ( f ) and P60 L4 SS cortex ( g ) of SRPX2 +/Y and SRPX2 −/Y mice. The total number of C1q/C3 particles are plotted as the mean ± s.e.m., and analyzed by two-tailed unpaired t test. For f (dLGN), C1q: SRPX2 +/Y , n = 6 from 3 mice, SRPX2 −/Y , n = 8 from 4 mice; C3: SRPX2 +/Y , n = 10, SRPX2 −/Y , n = 10, ** P = 0.0035, both from 5 mice. For g (L4 SS cortex), C1q: SRPX2 +/Y , n = 12 from 4 mice, SRPX2 −/Y , n = 9 from 3 mice; C3: SRPX2 +/Y , n = 9, SRPX2 −/Y , n = 9, ** P = 0.0080, both from 3 mice.

Article Snippet: Protein complexes were eluted with 100 μL of 3X FLAG peptide (200 ng/μL) according to the manufacturer’s protocol (Sigma-Aldrich), and the eluates were blotted with anti-FLAG M2-peroxidase (HRP) (Sigma-Aldrich, Cat. no. A8592-0.2mg), anti-C1Q (Abcam, Cat no. ab182451), and anti-C3 (MP Biomedicals, Cat. no. 0855730) antibodies.

Techniques: Inhibition, Activity Assay, Isolation, Transfection, Activation Assay, Two Tailed Test, Incubation, Negative Control, Positive Control, Staining, Quantitation Assay

Journal: PLoS ONE

Article Title: ADH IB Expression, but Not ADH III, Is Decreased in Human Lung Cancer

doi: 10.1371/journal.pone.0052995

Figure Lengend Snippet: 22.5 ng purified recombinant proteins were separated by SDS-PAGE, and immunoblots were performed to determine antibody reactivity to GSNOR, ADH IA, ADH IB, ADH II, and ADH IV. With the exception of ADH IA, the purified recombinant proteins were generated with a fusion protein tag during cloning, and the tag was cleaved off during purification as described in the . The molecular weights of the fusion proteins are approximately 50–51 kDa, and the final, purified proteins are 39–41 kDa. Human GSNOR consistently migrates faster than the calculated molecular weight. As seen by for the presence of both 50 and 40 kDa bands for ADH II, the majority of the protein in this preparation still contains the fusion protein tag. However, the purified GSNOR protein used for immunization was confirmed to be full length, and free of additional tag sequence as described in the . A) Commercially available rabbit polyclonal GSNOR antibody (Proteintech #11051-1-AP). B) In-house polyclonal antibody generated by immunization of rats with purified, recombinant, full length human GSNOR protein at Biomodels (Watertown, MA) for N30 Pharmaceuticals.

Article Snippet: Three distinct monoclonal antibodies to human GSNOR (N30-C3 rat anti-GSNOR, N30-F6 mouse anti-GSNOR, and N30-G11 mouse anti-GSNOR) were generated by immunization of mice or rats with purified, recombinant, full length human GSNOR protein at ProMab Biotechnologies (Richmond, CA) for N30 Pharmaceuticals.

Techniques: Purification, Recombinant, SDS Page, Western Blot, Generated, Cloning, Molecular Weight, Sequencing

Journal: PLoS ONE

Article Title: ADH IB Expression, but Not ADH III, Is Decreased in Human Lung Cancer

doi: 10.1371/journal.pone.0052995

Figure Lengend Snippet: Purified recombinant proteins were separated by SDS-PAGE, and immunoblots were performed to determine antibody reactivity to GSNOR, ADH IB, ADH II, and ADH IV. Three independent monoclonal antibodies were tested: A) N30-F6 mouse anti-GSNOR; B) N30-G11 mouse anti-GSNOR; C) N30-C3 rat anti-GSNOR.

Article Snippet: Three distinct monoclonal antibodies to human GSNOR (N30-C3 rat anti-GSNOR, N30-F6 mouse anti-GSNOR, and N30-G11 mouse anti-GSNOR) were generated by immunization of mice or rats with purified, recombinant, full length human GSNOR protein at ProMab Biotechnologies (Richmond, CA) for N30 Pharmaceuticals.

Techniques: Purification, Recombinant, SDS Page, Western Blot, Bioprocessing

Journal: PLoS ONE

Article Title: ADH IB Expression, but Not ADH III, Is Decreased in Human Lung Cancer

doi: 10.1371/journal.pone.0052995

Figure Lengend Snippet: Normal human lung tissue microarrays were stained with N30-C3 monoclonal GSNOR antibody (1 µg/mL) followed by DAB detection. Bronchial epithelial cells, alveolar macrophages, and type 2 pneumocytes in alveoli are strongly stained.

Article Snippet: Three distinct monoclonal antibodies to human GSNOR (N30-C3 rat anti-GSNOR, N30-F6 mouse anti-GSNOR, and N30-G11 mouse anti-GSNOR) were generated by immunization of mice or rats with purified, recombinant, full length human GSNOR protein at ProMab Biotechnologies (Richmond, CA) for N30 Pharmaceuticals.

Techniques: Staining

Journal: PLoS ONE

Article Title: ADH IB Expression, but Not ADH III, Is Decreased in Human Lung Cancer

doi: 10.1371/journal.pone.0052995

Figure Lengend Snippet: Normal human lung cancer tissue microarrays were stained with monoclonal GSNOR antibodies (N30-C3, N30-F6, N30-G11) or a commercially available polyclonal GSNOR antibody (11051-1-AP) followed by DAB detection. Various human lung cancer tissues are strongly stained by all three GSNOR monoclonal antibodies, but staining is less prominent when the non-specific polyclonal GSNOR antibody is used.

Article Snippet: Three distinct monoclonal antibodies to human GSNOR (N30-C3 rat anti-GSNOR, N30-F6 mouse anti-GSNOR, and N30-G11 mouse anti-GSNOR) were generated by immunization of mice or rats with purified, recombinant, full length human GSNOR protein at ProMab Biotechnologies (Richmond, CA) for N30 Pharmaceuticals.

Techniques: Staining, Bioprocessing