rabbit polyclonal anti c5a  (Bioss)

Journal: Investigative Ophthalmology & Visual Science

Article Title: Urban Particulate Matter Triggers Meibomian Gland Dysfunction

doi: 10.1167/iovs.65.2.8

Figure Lengend Snippet: Recruitment and activation of PMNs in UPM-treated mice. ( A ) Hematoxylin and eosin staining and ( B ) immunohistochemistry showed a lot of PMNs ( black arrows ) and CD45-positive cells surrounding the MG acini. ( C ) Immunohistochemistry and ( D ) immunofluorescence of NE disclosed significant upregulation in MGs from the UPM group compared with the control group. ( E – H ) Western blot analysis of ( F ) C5/C5a, ( G ) MMP-9 and ( H ) TNF-α suggested the expression levels of these proteins were all increased (all n = 4 mice per group). Scale bars:, 50 µm. Data are shown as mean ± SD. * p < 0.05 and *** p < 0.001.

Article Snippet: The membrane was then blocked in 3% BSA for 2 hours and immunoblotted with primary antibodies overnight: rabbit anti-HMGCR (1:1000, A16875), rabbit anti-SREBP-1 (1:1000, ab3259; Abcam), goat anti-Lrig1 (1:1000, AF3688), rabbit anti-K1 (1:1000, ab185628), rabbit anti-K10 (1:1000, ab76318), rabbit anti-cleaved caspase-8 (1:1000, 8592T; Cell Signaling Technology), rabbit anti-cleaved caspase-9 (1:1000, 20750S; Cell Signaling Technology), rabbit anti-cleaved caspase-3 (1:1000, 9664S), rabbit anti-NLRP3 (1:500, NBP2-12446), rabbit anti-caspase-1 and rabbit anti-cleaved caspase-1 (1:1000, A0964), rabbit anti-GSDMD (1:1000, A18281), rabbit anti-cleaved GSDMD (1:1000, ab209845; Abcam), rabbit anti-IL-18 (1:1000, A1115; Abclonal), rabbit anti-IL-1β (1:1000, ab234437; Abcam), rabbit anti-caspase-4 and rabbit anti-cleaved caspase-4 (1:1000, NBP3-13397; R&D Systems), rabbit anti-C5/C5a (1:1000, A8104; Abclonal), rabbit anti-matrix metalloproteinase-9 (MMP-9) (1:1000, A2095; Abclonal), rabbit anti-TNF-α (1:1000, A0277; Abclonal), rabbit anti-p65 (1:1000, 8242S; Cell Signaling Technology), rabbit anti-phospho-p65 (1:1000, 3033S; Cell Signaling Technology), rabbit anti-p38 (1:1000, 8690S; Cell Signaling Technology), rabbit anti-phospho-p38 (1:1000, 4511S; Cell Signaling Technology), rabbit anti-β-actin (1:2000, 4970S; Cell Signaling Technology), rabbit anti-GAPDH (1:2000, 5174S; Cell Signaling Technology).

Techniques: Activation Assay, Staining, Immunohistochemistry, Immunofluorescence, Western Blot, Expressing

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: Computational and Structural Biotechnology Journal

Article Title: Enzyme kinetic and binding studies identify determinants of specificity for the immunomodulatory enzyme ScpA, a C5a inactivating bacterial protease

doi: 10.1016/j.csbj.2021.04.024

Figure Lengend Snippet: Kinetic and thermodynamic parameters for ScpA S512A binding to C5a peptides.

Article Snippet: Western blot analysis of the samples used polyclonal rabbit anti-human C5a antibody (MBS524143, MyBioSource, USA).

Techniques: Binding Assay

Journal: Computational and Structural Biotechnology Journal

Article Title: Enzyme kinetic and binding studies identify determinants of specificity for the immunomodulatory enzyme ScpA, a C5a inactivating bacterial protease

doi: 10.1016/j.csbj.2021.04.024

Figure Lengend Snippet: ScpA enzyme kinetics and activity against rhC5a dR . (a) SDS-PAGE analysis of recombinant rhC5a and rhC5a dR cleaved with ScpA. Lanes with ‘+’ and ‘-’ indicate samples with and without ScpA, respectively. rhC5a and rhC5a dR treated with ScpA produced fragments of similar size (indicated with an arrow marked ‘core’). Molecular weight ladders are shown on the left side of the gels in Fig. 1 a and 1b in kDa. Mass spectrometry of cleaved rhC5a and rhC5a dR show that the observed mass of the products (10886.1 Da and 10885.6 Da, respectively) are consistent with rhC5a core (calculated mass 10886.2 Da.). Observed and calculated (parenthesis) masses are reported for rhC5a and rhC5a dR . (b) The activity of ScpA was examined in human serum. The Coomassie stained polyacrylamide gel and Western blot analysis shows that ScpA (36 nM) cleaves rhC5a (39 µM) in human serum. The arrow indicates the expected position for the ‘core’ (P N ) product of ScpA cleavage of C5a. (c) Progress curves for ScpA cleavage of rhC5a C75 -BODIPY. Data are plotted with curves from global fitting of 6 progress curves to the Van Slyke-Cullen model. Values for k cat and K m are reported as the mean and standard deviation of the mean from 3 experiments. Data shown in the plot are from 3 experiments. A key to substrate concentrations is provided on the right-hand side of the graph.

Article Snippet: Western blot analysis of the samples used polyclonal rabbit anti-human C5a antibody (MBS524143, MyBioSource, USA).

Techniques: Activity Assay, SDS Page, Recombinant, Produced, Molecular Weight, Mass Spectrometry, Staining, Western Blot, Standard Deviation

Journal: Computational and Structural Biotechnology Journal

Article Title: Enzyme kinetic and binding studies identify determinants of specificity for the immunomodulatory enzyme ScpA, a C5a inactivating bacterial protease

doi: 10.1016/j.csbj.2021.04.024

Figure Lengend Snippet: Model of the ScpA-hC5a complex. (a) The stereo diagram shows the ScpA-C5a complex with hC5a (cartoon diagram) on the surface of ScpA. The coordinates for the model are from the docking analysis described in Kagawa et al. . ScpA is colored by domains, with the catalytic domain (‘CAT’) domain in salmon, the PA domain in blue, and the Fn1–Fn3 domains in green, cyan, and yellow, respectively. hC5a is shown as a cartoon model with the core portion (P N , orange) on the ScpA Fn2 domain and tail residues (P C , purple) extended through the prime side of the active site. The location of the scissile bond is indicated with a red arrow. Sidechains of hC5a arginine residues found to impact on binding are shown with space filling models and labelled ‘a’ (R37), ‘b’ (R40), ‘c’ (R46), and ‘d’ (R74). Panel (b) shows a stereo diagram the hC5a model indicating locations of all residues mutated in this study (shown as stick models). hC5a is colored as in panel A. The four hC5a helices are labelled ‘I’ to ‘IV’. The location of the C27 sidechain is indicated with an asterisk. Panels (c) to (h) show representative sensorgrams of ScpA S512A binding to rhC5a K4A,K5A , rhC5a K12A,K14A , rhC5a R37A , rhC5a R40A , rhC5a R46A , and rhC5a K49A respectively. Observed data (black lines) are plotted with curves obtained from global fitting of data with a 1:1 Langmuir model for binding (red lines). Mean K D values obtained from 3 experiments are reported. Panel (i) shows a reaction scheme where binding of full-length hC5a (‘S’) occurs with a conformational change in ScpA (‘E’ to ‘F’). The C-terminal ‘tail’ residues (‘P C ’) are released during the acylation step. Deacylation produces a complex (‘F∙P N ’) between the enzyme and core portion of hC5a (‘P N ’). Dissociation of P N is faster from ‘F’ and thus not rate limiting in the catalytic cycle. Binding of the ‘P N ’ product to the ‘E’ ScpA state is accompanied by a slow rate of dissociation as measured in SPR studies. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Western blot analysis of the samples used polyclonal rabbit anti-human C5a antibody (MBS524143, MyBioSource, USA).

Techniques: Binding Assay

Journal: Frontiers in Immunology

Article Title: Complement C5a Induces Pro-inflammatory Microvesicle Shedding in Severely Injured Patients

doi: 10.3389/fimmu.2020.01789

Figure Lengend Snippet: C5a alters MV shedding in PMNs. (A) PMNs were stimulated for 0, 10, 30, 60, and 240 min with C5a (100 ng/ml) or LPS (5 μg/ml) and supernatants were analyzed by FACS. Untreated cells served as control. n = 10, One-Way ANOVA followed by Student-Neuman-Keuls post-hoc testing was performed for each time point, * p < 0.05. (B) MVs from supernatants were further analyzed for expression of C5aR1 by FACS. n = 3 per group, * p < 0.05. (C) Heat map and gene list of selected and differentially regulated genes upon C5a treatment in in neutrophils after 1 h ( n = 3 per group).

Article Snippet: After protein transfer, PVDF membranes were blocked in 5% milk/TBST and incubated with the primary antibody rabbit anti-human C5a (Calbiochem) overnight at 4°C.

Techniques: Control, Expressing

Journal: Frontiers in Immunology

Article Title: Complement C5a Induces Pro-inflammatory Microvesicle Shedding in Severely Injured Patients

doi: 10.3389/fimmu.2020.01789

Figure Lengend Snippet: Effect of the C5aR1-antagonist PMX53 on vesicle shedding. (A) Upper panel shows merged image of F-actin staining (red, Phalloidin-Alexa Flour 568) and nuclei staining (DAPI) of fixed PMNs, untreated, after treatment with native human C5a (100 ng/ml), and C5a treatment in combination with PMX53 (10 μM) for 1 h, respectively at 630x magnification. Lower panel with further zoom-in on PMNs from the upper panel, white arrows indicate shed MVs. Experiment was repeated at least three times. (B) Quantitative analysis of shed MV at different C5a concentrations and in presence of the PMX53 together and alone in 10 μl supernatant ( n = 3). * p < 0.05.

Article Snippet: After protein transfer, PVDF membranes were blocked in 5% milk/TBST and incubated with the primary antibody rabbit anti-human C5a (Calbiochem) overnight at 4°C.

Techniques: Staining

Journal: Frontiers in Immunology

Article Title: Complement C5a Induces Pro-inflammatory Microvesicle Shedding in Severely Injured Patients

doi: 10.3389/fimmu.2020.01789

Figure Lengend Snippet: Inflammatory feature of PMN-derived MVs. (A) One representative immunoblot probed for phospho-p47 phox of whole cell lysates from PMNs. PMNs were treated with C5a, C5a and PMX53, and PMX53 only. Untreated PMNs served as control. Afterwards, PMNs from autologous donors were incubated with the generated MVs for 1h at 37°C. PMNs without MV stimulation served as control. (B) Quantification of phospho-p47 phox signal intensity of PMNs whole cell lysates from three donors. * p < 0.05 compared to PMN without MV control. (C) Time-dependent ROS generation in PMNs incubated with MVs, n = 3, * p < 0.05. (D) MPO amount in PMN supernatants after incubation with MVs, n = 3 * p < 0.05. (E) C5a-gerenerated MVs were incubated in whole blood from autologous donors for 1 h and for 4 h, respectively. IL-6 was determined in plasma by ELISA. n = 5 per group, * p < 0.05.

Article Snippet: After protein transfer, PVDF membranes were blocked in 5% milk/TBST and incubated with the primary antibody rabbit anti-human C5a (Calbiochem) overnight at 4°C.

Techniques: Derivative Assay, Western Blot, Control, Incubation, Generated, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Complement C5a Induces Pro-inflammatory Microvesicle Shedding in Severely Injured Patients

doi: 10.3389/fimmu.2020.01789

Figure Lengend Snippet: Model for C5a-mediated alterations on MV shedding for neutrophils after PT. PT causes an early activation of complement generating increased C5a concentrations. C5a induces activity of the small GTPase Arf6, which is dependent on C5aR1 signaling. Additionally, specific changes in gene expression pattern are involved in MV shedding. Resting PMNs show physiological MV shedding and composition, which have no inflammatory potential. In contrast, PMNs incubated with C5a loose C5aR1 surface expression induced by C5aR1 signaling and Arf6-mediated shedding of C5aR1-positive MVs. Furthermore, these MVs show increased CD66b and C5aR1 expression, which show pro-inflammatory features when incubated with resting PMNs. C5a induces shedding of MVs, which lead to NAPDH oxidase activation, ROS generation and MPO release in resting PMNs. Moreover, IL-6 generation was observed when these MVs were incubated in whole blood. PMX53 and the Arf6-selective compound NAV2729 can block the shedding of C5aR1-positive MVs from PMN surfaces and its inflammatory effects.

Article Snippet: After protein transfer, PVDF membranes were blocked in 5% milk/TBST and incubated with the primary antibody rabbit anti-human C5a (Calbiochem) overnight at 4°C.

Techniques: Activation Assay, Activity Assay, Gene Expression, Incubation, Expressing, Blocking Assay