Journal: Molecular Therapy

Article Title: A Novel C5a-neutralizing Mirror-image ( l -)Aptamer Prevents Organ Failure and Improves Survival in Experimental Sepsis

doi: 10.1038/mt.2013.178

Figure Lengend Snippet: NOX-D20 binds to C5 but does not inhibit complement-mediated hemolysis. SPR measurement of NOX-D20 binding to human ( a ) C5a, ( b ) C5a(desArg), and ( c ) C5. Kinetic rate constants k a and k d are shown as mean ± SEM. Data are representative for at least three individual measurements. ( d ) Human serum pretreated with NOX-D20 (black squares) or C5-binding aptamer C5C6 (black triangles) was incubated with opsonized sheep erythrocytes. Hemolysis was quantified by photometric measurement of hemoglobin in the supernatant at 405 nm. Normalized data representative for three independent experiments is shown. RU, response units.

Article Snippet: Recombinant human and mouse C5a was from R&D Systems (Wiesbaden, Germany), recombinant human C5a(desArg) from Hycult Biotech (Beutelsbach, Germany), and human C5 purified from serum from Sigma-Aldrich (Taufkirchen, Germany).

Techniques: Binding Assay, Incubation

Journal: Frontiers in Medicine

Article Title: Selective Inhibition of Histone Deacetylase Class IIa With MC1568 Ameliorates Podocyte Injury

doi: 10.3389/fmed.2022.848938

Figure Lengend Snippet: Histone deacetylase IIa inhibition with MC1568 protects podocyte from injury in vitro . (A,B) Immunoblotting and quantification analysis of HDAC IIa members, desmin and α-SMA in ADR-stimulated podocytes with or without MC1568 treatment. (C) Phalloidin staining in ADR-stimulated podocytes with or without MC1568 treatment. Scale bar, 50 μm. Statistical analysis was performed with two-way ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Histone deacetylase 4 (Proteintech, 17449-1-AP, WB, 1:1,000, IHC, 1:100), HDAC5 (Proteintech, 16166-1-AP, WB, 1:1,000, IHC, 1:100), HDAC7 (Santa Cruz, sc-74563, WB, 1:1,000, IHC, 1:100), HDAC9 (Santa Cruz, sc-398003, WB, 1:1,000, IHC, 1:100), Desmin (Proteintech, 16520-1-AP, WB, 1:1,000, IHC), α-SMA (Proteintech, 14395-1-AP, WB, 1:1,000, IHC, 1:200), Active β-catenin (CST, 8814, WB, 1:3,000, IHC, 1:200), GAPDH (Proteintech, 10494-1-AP, WB, 1:10,000), β-tubulin (Proteintech, 10094-1-AP, WB, 1:10,000).

Techniques: Histone Deacetylase Assay, Inhibition, In Vitro, Western Blot, Staining

Journal: World Journal of Surgical Oncology

Article Title: Effects of bradykinin on the survival of multiterritory perforator flaps in rats

doi: 10.1186/s12957-019-1570-3

Figure Lengend Snippet: a Photographs of the postoperative flaps from the bradykinin and control groups on day 7. b The flap survival rate (%) in the bradykinin group (85.83 ± 0.98%) and control group (71.83 ± 2.52%). c The perfusion images of a flap on POD 7. Red denotes high perfusion, and blue denotes low perfusion with the scale bar. d The perfusion value on POD 7 (control group, 428.38 ± 23.39; bradykinin group, 505.85 ± 25.52). n = 5 per group. * P < 0.05, ** P < 0.01

Article Snippet: Rats in the bradykinin group were injected intraperitoneal bradykinin (150 μg/kg; purity = 98.97%; Medchem Express, Princeton, NJ, USA) [ ] 30 min before the procedure.

Techniques: Control

Journal: World Journal of Surgical Oncology

Article Title: Effects of bradykinin on the survival of multiterritory perforator flaps in rats

doi: 10.1186/s12957-019-1570-3

Figure Lengend Snippet: a Neovascularization in the bradykinin and control groups (original magnification × 100). b The percentage of microvascular density (MVD) in the bradykinin (39.47 ± 1.35/mm 2 ) and control (30.38 ± 2.10/mm 2 ) groups. n = 5 per group. * P < 0.05

Article Snippet: Rats in the bradykinin group were injected intraperitoneal bradykinin (150 μg/kg; purity = 98.97%; Medchem Express, Princeton, NJ, USA) [ ] 30 min before the procedure.

Techniques: Control

Journal: World Journal of Surgical Oncology

Article Title: Effects of bradykinin on the survival of multiterritory perforator flaps in rats

doi: 10.1186/s12957-019-1570-3

Figure Lengend Snippet: a The CD34-positive microvessels were represented by black arrows (original magnification × 100). b The number of CD34-positive vessels/mm 2 was 42.13 ± 2.59/mm 2 in the bradykinin group and 31.92 ± 1.40/mm 2 in the control group. n = 5 per group. * P < 0.05

Article Snippet: Rats in the bradykinin group were injected intraperitoneal bradykinin (150 μg/kg; purity = 98.97%; Medchem Express, Princeton, NJ, USA) [ ] 30 min before the procedure.

Techniques: Control

Journal: World Journal of Surgical Oncology

Article Title: Effects of bradykinin on the survival of multiterritory perforator flaps in rats

doi: 10.1186/s12957-019-1570-3

Figure Lengend Snippet: a SOD activity (U mg −1 protein −1 ). Bradykinin group, 45.46 ± 1.43; control group, 30.61 ± 1.47. b MDA content (nmol mg protein −1 ). Bradykinin group, 41.39 ± 1.67; control group, 60.05 ± 2.25. n = 5 per group. * P < 0.05

Article Snippet: Rats in the bradykinin group were injected intraperitoneal bradykinin (150 μg/kg; purity = 98.97%; Medchem Express, Princeton, NJ, USA) [ ] 30 min before the procedure.

Techniques: Activity Assay, Control

Journal: Frontiers in Immunology

Article Title: Interaction of Serum-Derived and Internalized C3 With DNA in Human B Cells—A Potential Involvement in Regulation of Gene Transcription

doi: 10.3389/fimmu.2019.00493

Figure Lengend Snippet: C3a in B cells is generated extracellularly by alternative pathway C3-convertases. Western blot results investigating the origin of C3a in Raji B cells. Cells were incubated with distinct sources of C3 (10% NHS, 100 μg/ml C3, 100 μg/ml C3met) in DGVB++, Mg-EGTA, or EDTA-GVB buffer for 1 h at 37 °C. To analyze complement pathway dependent generation of C3a, sera depleted in C1q (classical pathway), MBL (lectin pathway) or Factor B (alternative pathway) were used. To distinguish intracellular or C3-convertase dependent processing of C3, C3, and C3met were added alone (intracellular cleavage) or in the presence of C3-depleted serum (C3 convertase supplementation). C3 processing and C3a generation were analyzed by Western blot with the goat polyclonal anti-C3 (Quidel) and rabbit anti-C3a (Complement Technologies) antibodies under reducing conditions. Result shown is one representative out of three independent experiments.

Article Snippet: C3a was detected with the polyclonal rabbit anti-C3a antibody ( ) from Complement Technologies (#A218, Western blot) or with the monoclonal mouse anti-C3a/C3adesArg Ab from Hycult (#HM2074, gel shift assays and DNA ELISA).

Techniques: Generated, Western Blot, Incubation

Journal: Frontiers in Immunology

Article Title: Interaction of Serum-Derived and Internalized C3 With DNA in Human B Cells—A Potential Involvement in Regulation of Gene Transcription

doi: 10.3389/fimmu.2019.00493

Figure Lengend Snippet: C3 and C3a enter the nucleus after uptake. (A,B) Western blot results showing presence of C3 and C3a in nuclear compartments of Raji cells. The human B cell line, Raji was incubated with NHS, C3 or C3met as a source of C3 either in EDTA-GVB (A) or in Mg-EGTA (B) buffer for 1 h at 37°C. After lysis, cytoplasmic, soluble nuclear, and chromatin-associated nuclear fractions were separated and analyzed by Western blot with the goat polyclonal anti-C3 antibody under non reducing conditions (A) or with the rabbit polyclonal antibody against C3a under reducing condition (B) . The purity of distinct cellular fractions was verified using antibodies against B-actin (cytoplasmic marker), lamin B1 (nuclear marker) and histone H2B (chromatin-associated nuclear marker). Results shown are one representative experiment out of three (A) or four (B) independent analyzes. (C) Representative confocal images showing that AlexaFluor 488 labeled C3 enters the nucleus. 2 × 10 6 Raji cells were incubated with 100 μg/ml C3-AlexaFluor 488 for 30 min at 37°C, fixed and counterstained with DAPI using mounting medium. Representative images are shown from two independent experiments investigating at least 50 cells/analysis.

Article Snippet: C3a was detected with the polyclonal rabbit anti-C3a antibody ( ) from Complement Technologies (#A218, Western blot) or with the monoclonal mouse anti-C3a/C3adesArg Ab from Hycult (#HM2074, gel shift assays and DNA ELISA).

Techniques: Western Blot, Incubation, Lysis, Marker, Labeling

Journal: Frontiers in Immunology

Article Title: Interaction of Serum-Derived and Internalized C3 With DNA in Human B Cells—A Potential Involvement in Regulation of Gene Transcription

doi: 10.3389/fimmu.2019.00493

Figure Lengend Snippet: C3 and C3a bind to distinct types of DNA. (A–C) Gel shift assays presenting interaction between C3(a) and genomic DNA isolated from Raji cells. Isolated genomic DNA was incubated either with pre-titrated concentrations of C3 (A) or C3a (B) or C3b (C) and separated by agarose gel electrophoresis. Formation of high molecular weight DNA-protein complexes is indicated by slower DNA migration (DNA shift) compared to migration of the free nucleic acid. The presence of C3 and its cleavage fragments in the pre-formed protein-DNA complexes was confirmed with anti-C3a and anti-C3d antibodies caused supershift. One representative experiment out of five performed is shown. (D) Gel shift assays showing that C3(a)—DNA interaction is not ionic in nature. C3 or C3a were incubated with genomic DNA of Raji cells in the presence of increasing NaCl concentration (ranging from 150 to 1,200 mM). Binding of C3 and C3a to DNA was observed at 8X higher salt concentration than the physiological 150 mM. (E) Interaction of C3 and C3a with DNA is not restricted to genomic DNA. Linearized pCEP4 vector was incubated with either C3 or C3a and DNA-protein complex formation was investigated by gel shift assay. Results illustrated are one representative out of three independent experiments. (F,G) ELISA results confirming interaction between C3(a) and DNA. ELISA microplate surfaces were coated either with DNA (F) or C3, C3a, C3b (G) . After incubation with distinct concentrations of C3 proteins (F) or Raji genomic DNA (G) , protein-DNA interactions were detected using anti-C3d, anti-C3a (anti-C3 ELISA, F ), or anti-dsDNA antibody (anti-DNA ELISA, G ). Results shown are mean absorbance values measured at 490 nm with SD of four independent experiments. (H) ELISA results showing presence of C3-DNA complexes in formaldehyde cross-linked chromatin fraction of Raji B cells. Cells were incubated in the presence or absence of C3, C3met, fixed with 1% formaldehyde to cross-link protein-DNA complexes and chromatin isolated using commercial kit. C3-DNA complexes were investigated by ELISA on anti-C3 or anti-DNA coated plates. Data are shown as mean absorbance values measured at 490 nm with SD of three independent experiments. Differences with p < 0.05 were considered statistically significant and compared to non-treated cells (two-way ANOVA with Dunnett's multiple comparison, ns p > 0.05, * p < 0.05, ** p < 0.01).

Article Snippet: C3a was detected with the polyclonal rabbit anti-C3a antibody ( ) from Complement Technologies (#A218, Western blot) or with the monoclonal mouse anti-C3a/C3adesArg Ab from Hycult (#HM2074, gel shift assays and DNA ELISA).

Techniques: Electrophoretic Mobility Shift Assay, Isolation, Incubation, Agarose Gel Electrophoresis, Molecular Weight, Migration, Concentration Assay, Binding Assay, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Interaction of Serum-Derived and Internalized C3 With DNA in Human B Cells—A Potential Involvement in Regulation of Gene Transcription

doi: 10.3389/fimmu.2019.00493

Figure Lengend Snippet: Interaction of C3 and its cleavage fragments with histones. Recombinant histone core octamers (H2A/H2B/H3/H4), the linker H1 histone, or alpha-1-antitrypsin (negative control) were immobilized on microtiter plates and incubated with increasing concentrations of C3 (A) , C3b (B) , or C3a (C) . As negative control, BSA was used. Binding was detected either with monoclonal anti-C3d (A,B) or polyclonal anti-C3a (C) antibodies. Background signals obtained from BSA incubation were subtracted from the original values. Data are presented as mean absorbance values measured at 490 nm with SD of three independent experiments. Differences with p < 0.05 were considered statistically significant and compared to 0 μg/ml protein incubated surfaces (two-way ANOVA with Dunnett's multiple comparison, ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Upper asterisks indicate statistical significance of H1-, lower asterisks of histone octamer coated surfaces compared to binding to A1AT. (D) gDNA of Raji cells was coated on microtiter plates and AlexaFluor 488-labeled H1 histone binding was monitored in the presence or absence of C3a. As negative control, BSA was used. Background signals obtained on A1AT coated surfaces were subtracted from the original values. Data are presented as relative fluorescent unit (RFU) measured at 525 nm with SD of two independent experiments. Differences with p < 0.05 were considered statistically significant and compared to no C3a treated samples (two-way ANOVA with Dunnett's multiple comparison, ns p > 0.05, * p < 0.05, ** p < 0.01). Upper and lower asterisks indicate statistical significance of H1-AlexaFluor 488 binding in the presence of BSA or C3a (respectively) compared to binding of H1-AlexaFluor 488 alone.

Article Snippet: C3a was detected with the polyclonal rabbit anti-C3a antibody ( ) from Complement Technologies (#A218, Western blot) or with the monoclonal mouse anti-C3a/C3adesArg Ab from Hycult (#HM2074, gel shift assays and DNA ELISA).

Techniques: Recombinant, Negative Control, Incubation, Binding Assay, Labeling

Journal: Journal of clinical medicine

Article Title: Serum Desmosine Levels Might Be Associated with the Size of Ruptured Cerebral Aneurysms in Patients with Aneurysmal Subarachnoid Hemorrhage-A Preliminary Study.

doi: 10.3390/jcm14062056

Figure Lengend Snippet: Figure 1. Serum desmosine levels in the patient group (n = 135) and the control group (n = 25). Ctrl, control, ***, p < 0.001.

Article Snippet: For the desmosine measurement, a human desmosine ELISA kit was used (Cusabio, Houston, TX, USA, CSB-E12871h).

Techniques: Control

Journal: Journal of clinical medicine

Article Title: Serum Desmosine Levels Might Be Associated with the Size of Ruptured Cerebral Aneurysms in Patients with Aneurysmal Subarachnoid Hemorrhage-A Preliminary Study.

doi: 10.3390/jcm14062056

Figure Lengend Snippet: Figure 2. Serum desmosine levels collected 24 h after rupture, categorized by the size of the aneurysm measured on 3D-DSA. The size-based categorization of aneurysms is based on the PHASES score [4] classification. ns: non-significant; number of aneurysms in each group: <7 mm (n = 69), 7–9.9 mm (n = 25), 10–19.9 mm (n = 41). ***, p < 0.001, ****, p < 0.0001.

Article Snippet: For the desmosine measurement, a human desmosine ELISA kit was used (Cusabio, Houston, TX, USA, CSB-E12871h).

Techniques:

Journal: Journal of clinical medicine

Article Title: Serum Desmosine Levels Might Be Associated with the Size of Ruptured Cerebral Aneurysms in Patients with Aneurysmal Subarachnoid Hemorrhage-A Preliminary Study.

doi: 10.3390/jcm14062056

Figure Lengend Snippet: Figure 3. ROC curve for serum desmosine levels in detecting aneurysms larger than 7 mm. The best cut-off value was 0.533 ng/mL with the sensitivity and specificity of 81.7% and 60.3%, respectively. Red dashed line: line of equality or random chance.

Article Snippet: For the desmosine measurement, a human desmosine ELISA kit was used (Cusabio, Houston, TX, USA, CSB-E12871h).

Techniques:

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Activation of angiogenin expression in macrophages by lipopolysaccharide via the TLR4/NF-κB pathway in colitis

doi: 10.3724/abbs.2024013

Figure Lengend Snippet: Table 1 Sequences of primers and siRNAs used in this study

Article Snippet: The membrane was blocked with 5% nonfat milk and then incubated with primary antibodies, including anti-ANG antibody (prepared in our own laboratory) and anti-ACTB antibody (#81115-1-RR; Proteintech, Chicago, USA), in TBST (Tris-buffered saline, 0.1% Tween 20) buffer at 4°C overnight.

Techniques: Negative Control

Journal: Immunology

Article Title: Development and characterization of novel anti‐C5 monoclonal antibodies capable of inhibiting complement in multiple species

doi: 10.1111/imm.13083

Figure Lengend Snippet: Functional assays to determine whether monoclonal antibodies (mAb) 4G2, 7D4 and 10B6 inhibit complement in different species. (a–e) Classical pathway haemolysis (CH50). Sera tested were human (a), rat (b), rabbit (c), guinea pig (d) and mouse (e). Commercial mAb RO7112689 and Eculizumab were used as comparators. (f) Calculation of 50% inhibitory dose showed that human C5 inhibition by mAb 10B6 was equivalent to the two comparator mAb, RO7112689 and Eculizumab, and that 4G2 and 7D4 strongly inhibited rat C5. (g, h) Alternative pathway (AP) haemolysis (AH 50 ) assay using human (g) and rat (h) serum; all tested mAb inhibited AP in human serum, while 4G2 and 7D4 inhibited rat AP. (i) Inhibition of C5a generation by the novel mAb in a classical pathway assay with human serum; all three mAb efficiently inhibited C5a generation in a dose‐dependent manner. All experiments were repeated three times with the same results. The error bars are standard errors of triplicates. The dashed lines correspond to the comparator mAb.

Article Snippet: Blots were probed with anti‐C5a mAb (#HM2079; Hycult Biotech), detected using donkey anti‐mouse IgG‐HRP (Jackson ImmunoResearch, 715‐035‐150).

Techniques: Functional Assay, Inhibition

Journal: Immunology

Article Title: Development and characterization of novel anti‐C5 monoclonal antibodies capable of inhibiting complement in multiple species

doi: 10.1111/imm.13083

Figure Lengend Snippet: Impact of anti‐C5 monoclonal antibodies (mAb) on cleavage of C5 by neutrophil elastase. (a) Western blot of atypical cleavage of C5 by neutrophil elastase (NE). C5 was mixed with each mAb at 5× molar excess in HEPES‐buffered saline (HBS), NE was added at 420 n m , incubated and the reaction was stopped by addition of protease inhibitors. Samples (1 μg) were resolved on 4–20% SDS–PAGE gels under reducing (R) conditions and processed for WB to detect intact or cleaved C5 and C5a using an anti‐C5a mAb that also detects native C5 α chain (Hycult; mAb 2942). C5 α (115 000 MW), C5a (10·4 000 MW), CI; proteases cocktail inhibitors, Ecul.; Eculizumab, RO; RO7112689, 3D3; irrelevant non‐blocking anti‐C5 mAb. Note that numerous unidentified C5 cleavage products are present in all NE‐treated samples. (b) Densitometry analysis of the C5a band using imagej (expressed as % relative to the C5a generation in the absence of antibody; 100%) confirmed that mAb 4G2 and commercial mAb RO7112689 efficiently inhibited generation of C5a. (c) measurement of C5a generation by ELISA confirmed that mAb 4G2 and RO7112689 inhibited C5a generation while other mAb inhibited weakly. ELISA results are presented as percentage relative to C5a generation in the absence of any mAb (set as 100%; measured as 670 ng/ml in the ELISA). Results are representative of three independent experiments. The error bars are standard errors of triplicates.

Article Snippet: Blots were probed with anti‐C5a mAb (#HM2079; Hycult Biotech), detected using donkey anti‐mouse IgG‐HRP (Jackson ImmunoResearch, 715‐035‐150).

Techniques: Western Blot, Incubation, SDS Page, Blocking Assay, Enzyme-linked Immunosorbent Assay