Journal: Virulence

Article Title: Streptococcus suis subtilisin-like serine proteases SspA-1 and SspA-2 interplay with complement C3a and C5a to facilitate bacterial immune evasion and infection.

doi: 10.1080/21505594.2023.2301246

Figure Lengend Snippet: Figure 4. S. suis strains at stationary phase (4 × 106 CFU) were incubated with GST_C3a_GFP (0.5 μg) or GST_C5a_GFP (0.5 μg) in TSB for 6, 12 and 16 hours. Protease inhibitor cocktail in GST_C3a_GFP or GST_C5a_GFP without S. suis strains was used as a negative control. Monoclonal antibodies specific for C3 or C5 were applied for C3a and C5a immunodetection, respectively.

Article Snippet: Mouse anti-C5 monoclonal antibody (Proteintech, Wuhan, China) was applied at 37 °C for 1 hour to test the C5a binding capacity.

Techniques: Incubation, Protease Inhibitor, Negative Control, Bioprocessing, Immunodetection

Journal: Scientific Reports

Article Title: C5a–C5AR1 axis as a potential trigger of the rupture of intracranial aneurysms

doi: 10.1038/s41598-024-53651-7

Figure Lengend Snippet: The co-expression of C5AR1 and its ligand C5a in intracranial aneurysm lesions. Specimens of intracranial aneurysm (IA) lesions (Aneurysm) or control arterial walls (Superficial temporal artery in ( A ), Anterior cerebral artery-olfactory artery bifurcation in ( B )) from human cases ( A ) or rats ( B ) were harvested and examined in immunohistochemistry. The representative images of immunofluorescent staining of IA lesions for C5AR1 (green), C5a/C5a des-arg (red in ( A )), C5 (red in ( B )), and merged images with nuclear staining by DAPI (blue) are shown. A demagnified image of lower panels in ( B ) is shown on the right. The square in the demagnified image corresponds to the area shown in the magnified images. Scale bar: 50 μm.

Article Snippet: Primary antibodies used were as follows; rabbit polyclonal anti-CD88 (C5AR1) antibody (#PAB26140, Abnova, Taipei, Taiwan), mouse monoclonal anti-CD31 antibody (clone JC70A, #M0823, Dako, Glostrup, Denmark), mouse monoclonal anti-smooth muscle α-actin antibody (clone 1A4, #14-9760-82, Thermo Fisher Scientific, Waltham, MA, USA), rabbit polyclonal anti-myeloperoxidase antibody (#ab9535, abcam, Cambridge, UK), mouse monoclonal anti-human C5a/C5a des-Arg antibody (clone 2952, #HM2079, Hycult Biotech, Wayne, PA, USA), rabbit polyclonal anti-tPA antibody (#10147-1-AP, Proteintech Group, Rosemont, IL, USA), and rabbit polyclonal anti-plasminogen antibody (#GTX102877, Gene Tex, Irvine, CA, USA).

Techniques: Expressing, Control, Immunohistochemistry, Staining

Journal: Scientific Reports

Article Title: C5a–C5AR1 axis as a potential trigger of the rupture of intracranial aneurysms

doi: 10.1038/s41598-024-53651-7

Figure Lengend Snippet: Tissue-type Plasminogen Activator as an up-stream factor to produce C5a from C5. ( A ) The expression of tissue-type Plasminogen Activator (tPA) and Plasminogen in intracranial aneurysm (IA) lesions. IA lesions from human cases or induced in rats were harvested subjected to immunohistochemical analyses. The images of immunofluorescent staining of IA lesions for tPA (green), Plasminogen (red), and merged images with nuclear staining by DAPI (blue) are shown. Scale bar: 50 μm. ( B ) Co-expression of tPA with C5a in IA lesions. Human IA lesions were harvested subjected to immunohistochemical analyses. The images of immunofluorescent staining of IA lesions for C5a/C5a des-arg which is the derivative of C5a (green), tPA (red), and merged images with nuclear staining by DAPI (blue) are shown. Scale bar: 50 μm. ( C ) Enzymatic cleavage of C5 into C5a by Plasmin. Recombinant C5 (100 μg/ml) was co-incubated with each dose of recombinant Plasmin as indicated (0–100 μg/ml) for 1.5 h in a cell-free system. The incubation mixture was subjected to western blot analyses. The representative images from the western blot analyses to detect C5 or C5a are shown. M; size marker. ( D ) The alternation in the coagulation-fibrinolysis system in IA lesions of rats. The heat map of genes related with the coagulation-fibrinolysis system from the comprehensive gene expression profile data set (GEO accession: GSE161044) is shown.

Article Snippet: Primary antibodies used were as follows; rabbit polyclonal anti-CD88 (C5AR1) antibody (#PAB26140, Abnova, Taipei, Taiwan), mouse monoclonal anti-CD31 antibody (clone JC70A, #M0823, Dako, Glostrup, Denmark), mouse monoclonal anti-smooth muscle α-actin antibody (clone 1A4, #14-9760-82, Thermo Fisher Scientific, Waltham, MA, USA), rabbit polyclonal anti-myeloperoxidase antibody (#ab9535, abcam, Cambridge, UK), mouse monoclonal anti-human C5a/C5a des-Arg antibody (clone 2952, #HM2079, Hycult Biotech, Wayne, PA, USA), rabbit polyclonal anti-tPA antibody (#10147-1-AP, Proteintech Group, Rosemont, IL, USA), and rabbit polyclonal anti-plasminogen antibody (#GTX102877, Gene Tex, Irvine, CA, USA).

Techniques: Expressing, Immunohistochemical staining, Staining, Recombinant, Incubation, Western Blot, Marker, Coagulation

Journal: Scientific Reports

Article Title: C5a–C5AR1 axis as a potential trigger of the rupture of intracranial aneurysms

doi: 10.1038/s41598-024-53651-7

Figure Lengend Snippet: C5a–C5AR1 signaling cascade-dependent induction of pro-inflammatory factors. ( A – C ) The C5a–C5AR1 signaling cascade-dependent induction of pro-inflammatory genes in phorbol myristate acetate-primed cell line from human case with diffuse histiocytic lymphoma, U937 cell line. Cells were treated with recombinant C5a as indicated in the panels (0–10 h in ( A ), 0–100 nM in ( B ), 10 nM and 10 h in ( C ). Some cells were pre-treated with the selective C5AR1 inhibitor, W54011 (300 nM, 30 min) prior to the treatment with recombinant C5a. Total RNA was then purified from treated cells subjected to quantitative RT-PCR analyses to examine the induction of TNF (a gene for TNF-α), IL1B or PTGS2 (a gene for COX-2; cyclooxygenase-2). Data represents as mean ± s.e.m (( A ), n = 4; ( B , C ), n = 6). Statistical analysis was done by the Wilcoxon rank sum test in ( A ), the Kruskal–Wallis test followed by the Steel–Dwass test in ( B , C ). *p < 0.05. ( D ) Co-expression of C5a with pro-inflammatory factors in intracranial aneurysm (IA) lesions. IA lesions induced in rats were harvested subjected to immunohistochemical analyses. The images of immunofluorescent staining of IA lesions for C5ar1 (green), TNF-α (red), IL-1β (red), COX-2 (red), and merged images with nuclear staining by DAPI (blue) are shown. Scale bar: 50 μm.

Article Snippet: Primary antibodies used were as follows; rabbit polyclonal anti-CD88 (C5AR1) antibody (#PAB26140, Abnova, Taipei, Taiwan), mouse monoclonal anti-CD31 antibody (clone JC70A, #M0823, Dako, Glostrup, Denmark), mouse monoclonal anti-smooth muscle α-actin antibody (clone 1A4, #14-9760-82, Thermo Fisher Scientific, Waltham, MA, USA), rabbit polyclonal anti-myeloperoxidase antibody (#ab9535, abcam, Cambridge, UK), mouse monoclonal anti-human C5a/C5a des-Arg antibody (clone 2952, #HM2079, Hycult Biotech, Wayne, PA, USA), rabbit polyclonal anti-tPA antibody (#10147-1-AP, Proteintech Group, Rosemont, IL, USA), and rabbit polyclonal anti-plasminogen antibody (#GTX102877, Gene Tex, Irvine, CA, USA).

Techniques: Recombinant, Purification, Quantitative RT-PCR, Expressing, Immunohistochemical staining, Staining

Journal: Biology

Article Title: Plasma Proteomic Changes of Atherosclerosis after Exercise in ApoE Knockout Mice

doi: 10.3390/biology11020253

Figure Lengend Snippet: The differentially proteins between WD and WD EX groups.

Article Snippet: Then, the sections were incubated overnight at 4 °C with primary rabbit monoclonal antibody against complement C5 (PA2308, Boster, CA, USA, 1:1000) and then horseradish peroxidase–conjugated secondary antibody was used.

Techniques: Protease Inhibitor, Coagulation

Journal: Biology

Article Title: Plasma Proteomic Changes of Atherosclerosis after Exercise in ApoE Knockout Mice

doi: 10.3390/biology11020253

Figure Lengend Snippet: Effects of exercise on ( a ) aortic complement factor C5 expression and ( b ) quantitative analysis of the complement C5 IOD/area in ApoE knockout mice. Quantification of complement C5 staining and representative images. DAB-specific threshold selection (in red) from selected aortic root areas was performed using ImageJ, and total selective area was quantified and statistically analyzed. One-way ANOVA followed by Tukey’s post hoc test was used for statistical analysis, and *** p < 0.001 represents the significance between ND and WD groups. ## p < 0.01 represents the significance between WD and WD EX groups. n.s. = No significant difference.

Article Snippet: Then, the sections were incubated overnight at 4 °C with primary rabbit monoclonal antibody against complement C5 (PA2308, Boster, CA, USA, 1:1000) and then horseradish peroxidase–conjugated secondary antibody was used.

Techniques: Expressing, Knock-Out, Staining, Selection

Journal: PLoS ONE

Article Title: Complement inhibition attenuates acute kidney injury after ischemia-reperfusion and limits progression to renal fibrosis in mice

doi: 10.1371/journal.pone.0183701

Figure Lengend Snippet: (A) Immunoblotting analysis for C5a in renal tissue at 24 and 72 hours post-IRI. (B) Quantification of C5a band density from western blots normalized to β-tubulin. Results are expressed as mean ± SD fold changes (n = 3 for each group) compared with native controls. (C) Immunoblotting analysis of phosphorylated ERK1/2 normalized to total ERK1/2 (p44/42 MAPK) in renal tissue at 24 and 72 hours post-IRI in renal tissue. (D) Quantification of phosphorylated ERK band density from western blots normalized total ERK and β-tubulin. Results are expressed as mean ± SD fold changes (n = 3 for each group) compared with native controls. (E) Representative images of immunofluorescent microscopy performed on renal tissue at 72 hours post-injury targeting C3b deposition (200X). (F) mRNA expression of bradykinin 1 (BR1) and BR2 receptor in renal tissue. Expression was normalized to baseline expression of native controls and GAPDH was used as the endogenous control. Results are expressed as mean ± SD fold changes (n = 3 for each group).

Article Snippet: We used monoclonal rat anti-mouse C5a antibody (RD systems) and the phosphorylated and non-phosphorylated forms of ERK1/2 were detected using rabbit anti-mouse p44/42 MAPK polyclonal antibody (Cell Signaling).

Techniques: Western Blot, Microscopy, Expressing, Control