Journal: PLoS ONE

Article Title: Nanoparticulate matter exposure results in neuroinflammatory changes in the corpus callosum

doi: 10.1371/journal.pone.0206934

Figure Lengend Snippet: (A) Filtered air (n = 8) or nPM (n = 8) exposed mice stained for C5 (red) in the corpus callosum. Nuclei (DAPI) are stained in blue (400x). (B) Low magnification representation of region analyzed. (C) C5 immunostaining in the corpus callosum was significantly higher in nPM exposed animals compared to the filtered air group (p = 0.001). (D) Filtered air (n = 8) and nPM (n = 8) exposed mice stained for C5α (red) in the corpus callosum. Nuclei (DAPI) are stained in blue (400x). (E) Low magnification representation of region analyzed. (F) C5α immunostaining in the corpus callosum of nPM exposed animals was significantly greater than in the filtered air group (p = 0.02). (G) Filtered air (n = 18) and nPM (n = 18) exposed mice stained for CD88 (red) in the corpus callosum. Nuclei (DAPI) are stained in blue (200x). (H) Low magnification representation of region analyzed. (I) CD88 immunostaining in the corpus callosum was significantly higher in nPM exposed animals compared to the filtered air group (p = 0.04). * signifies p< 0.05, ** signifies p ≤ 0.001. Error bars represent standard deviation. Scale bars indicate 50 μm.

Article Snippet: Slides were incubated overnight with anti-C5 (mouse 1:50 Hycult Biotech, Netherlands; clone BB5.1), anti-CD88 (rat 1:200 Hycult Biotech, Netherland; HM1076) or rabbit complement component C5α (125kDa) antibody (1:50 Santa Cruz, SC-21941).

Techniques: Staining, Immunostaining, Standard Deviation

Journal: Investigative Ophthalmology & Visual Science

Article Title: Modulation of Retinal M?ller Cells by Complement Receptor C5aR

doi: 10.1167/iovs.13-12428

Figure Lengend Snippet: Detection of C5aR transcripts in both primary Müller cells and MIO-M1 cells. Total RNA isolated from primary Müller cells and MIO-M1 cells was reverse transcribed and used as a template to amplify a fragment of approximately 450 bp within the C5aR transcript spanning different exons. RNA that had not undergone reverse transcription was used as a negative control. Cp, primary cell control; Cm, MIO-M1 control. Marker: 100-bp DNA ladder.

Article Snippet: After this step, the cells were washed with 1 mL FACS buffer (PBS plus 5% bovine serum albumin), then stained with a monoclonal rabbit anti-human C5aR antibody (BD Biosciences, San Jose, CA) or the same concentration of rabbit IgG, followed by a second staining with FITC-conjugated goat anti-rabbit IgGs (5 μg/mL).

Techniques: Isolation, Negative Control, Marker

Journal: Investigative Ophthalmology & Visual Science

Article Title: Modulation of Retinal M?ller Cells by Complement Receptor C5aR

doi: 10.1167/iovs.13-12428

Figure Lengend Snippet: C5aR expression in Müller cells is upregulated by PGE2 as examined by qRT-PCR (A), flow cytometry (B), and confocal microscopy (C). (A) Quantitative RT-PCR assessment of C5aR expression in Müller cells after PGE2 stimulation. Müller cells were treated with 1 μM PGE2, and total RNAs were isolated after 0, 6, and 24 hours of PGE2 stimulation. C5aR transcript levels were measured by qRT-PCR and normalized against 18S RNA levels. (B) Flow analysis of C5aR expression on surface of Müller cells. C5aR was barely detectable in both primary cells (upper panel) and MIO-M1 cells (lower panel) under normal culture conditions (without PGE2); C5aR expression was increased and detectable in both types of cells after incubation with 1 μM PGE2 for 24 hours (with PGE2). Dotted line, isotype control; solid line, anti-C5aR mAb staining. Representative results from three individual experiments. (C) Confocal analysis of C5aR expression on Müller cells, showing results comparable to those from flow cytometry analysis.

Article Snippet: After this step, the cells were washed with 1 mL FACS buffer (PBS plus 5% bovine serum albumin), then stained with a monoclonal rabbit anti-human C5aR antibody (BD Biosciences, San Jose, CA) or the same concentration of rabbit IgG, followed by a second staining with FITC-conjugated goat anti-rabbit IgGs (5 μg/mL).

Techniques: Expressing, Quantitative RT-PCR, Flow Cytometry, Confocal Microscopy, Isolation, Incubation, Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Modulation of Retinal M?ller Cells by Complement Receptor C5aR

doi: 10.1167/iovs.13-12428

Figure Lengend Snippet: Hyperglycemia upregulates C5aR expression on Müller cells. (A) MIO-M1 cells were cultured in high-glucose (30 mM) media for 7 days with daily media change, then cells were cultured in the absence or presence of 1 μM PGE2 for another 24 hours, followed by flow cytometry analysis of C5aR expression on the surface of Müller cells. Dotted line, isotype control; solid line, anti-C5aR mAb staining. (B) MIO-M1 cells were cultured in normal (5 mM) or high-glucose (30 mM) media for 7 days with daily media change, then cultured in the absence or presence of 1 μM PGE2 for another 24 hours. Then different amounts of C5a, with or without 50 μM C5aRA, were added into each well. Forty-eight hours later, IL-6 and VEGF levels in the culture supernatants were measured by conventional ELISA. *P < 0.05.

Article Snippet: After this step, the cells were washed with 1 mL FACS buffer (PBS plus 5% bovine serum albumin), then stained with a monoclonal rabbit anti-human C5aR antibody (BD Biosciences, San Jose, CA) or the same concentration of rabbit IgG, followed by a second staining with FITC-conjugated goat anti-rabbit IgGs (5 μg/mL).

Techniques: Expressing, Cell Culture, Flow Cytometry, Staining, Enzyme-linked Immunosorbent Assay

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:

Article Title: Modulation of C5aR Expression on Human Neutrophils by Encapsulated and Acapsular Cryptococcus neoformans

doi: 10.1128/IAI.70.7.3363-3370.2002

Figure Lengend Snippet: Cytofluorimetric analysis of C5aR expression on PMN. PMN were incubated for 30 min in the absence (black histograms) or in the presence (grey lines) of (i) heat-killed acapsular cryptococci (7698) (E/T ratio, 1:4) (B), (ii) live 7698 (E/T ratio, 1:4) (C), (iii) heat-killed encapsulated cryptococci (6995) (E/T ratio, 1:4) (D), or (iv) live 6995 (E/T ratio, 1:4) (E). (A) Control showing autofluorescence of the cells (dotted line) and staining profile with an irrelevant antibody (black line). The results are from one experiment representative of four with similar results, performed by using cells from four different healthy subjects. FL1-H, fluorescent channel 1 height.

Article Snippet: Rabbit anti-human CD88 (C5aR) immunoglobulin G (IgG) was purchased from Serotec Ltd. (Oxford, England).

Techniques: Expressing, Incubation, Staining

Journal:

Article Title: Modulation of C5aR Expression on Human Neutrophils by Encapsulated and Acapsular Cryptococcus neoformans

doi: 10.1128/IAI.70.7.3363-3370.2002

Figure Lengend Snippet: Cytofluorimetric analysis of C5aR expression on PMN with GXM. PMN were incubated for 30 min in the absence (black histograms) or in the presence (grey lines) of (i) heat-killed acapsular cryptococci (7698) (E/T ratio, 1:4) (B), (ii) GXM alone (250 μg/ml) (C), or (iii) 7698 cells (E/T ratio, 1:4) pretreated with GXM (D). (A) Control showing autofluorescence of the cells (dotted line) and staining profile with an irrelevant antibody (black line). The results are from one experiment representative of four with similar results, performed by using cells from four different healthy subjects. (E) Mean fluorescence intensity for all groups. The results are the mean and standard error of the mean for four independent experiments with cells from four different healthy subjects. Statistical significance was determined by using ANOVA with Bonferroni's posttest analysis. FL1-H, fluorescent channel 1 height.

Article Snippet: Rabbit anti-human CD88 (C5aR) immunoglobulin G (IgG) was purchased from Serotec Ltd. (Oxford, England).

Techniques: Expressing, Incubation, Staining, Fluorescence

Journal: Journal of Biological Chemistry

Article Title: Third Extracellular Loop (EC3)-N Terminus Interaction Is Important for Seven-transmembrane Domain Receptor Function

doi: 10.1074/jbc.m110.129213

Figure Lengend Snippet: FIGURE 1. Sequence alignment of CXCR4 and C5aR with rhodopsin highlighting the most conserved residues and the known activation switches in GPCRs. Ballesteros and Weinstein nomenclature assigns X.50, to the “fingerprint residues” shown as white letters with black background in each transmembrane domain. Important transmembrane activation domains are shaded in gray. Conserved cysteine residues par- ticipatingindisulfidebondformationareshownwithgrayboxes.Residuesshownwithblackboxesindicatelack of complementary charged residues at 6.30 to support the ionic lock activation switch. Point mutation known to confer constitutive activity in CXCR4 is shown with white box. Residues shown as underlined have been subjected to mutagenesis for engineering C5aR. Residues marked with stars have been mutated to cysteine in other studies for engineering an extra disulfide bond between EC3 and N terminus of rhodopsin.

Article Snippet: The emulsion was heated for 10 min at 50 °C, cooled on ice, and centrifuged for 5 min. 25 l of each supernatant was resolved on a NuPAGE Novex 4–12% BisTris gel, transferred to nitrocellulose membrane, and immunoblotted with a rabbit polyclonal anti-C5aR antibody raised against full-length endogenous C5aR (Novus Biologicals, Littleton, CO).

Techniques: Sequencing, Activation Assay, Mutagenesis, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Third Extracellular Loop (EC3)-N Terminus Interaction Is Important for Seven-transmembrane Domain Receptor Function

doi: 10.1074/jbc.m110.129213

Figure Lengend Snippet: FIGURE 3. Engineering C5aR to a chemokine-type receptor; signaling profile of mutant C5aR receptors, co-expressed with C5a in yeast. Left panel, canonical receptor topology for C5aR. The N119S mutation on TM3isanovelconstitutivelyactivemutantidentifiedforC5aRinthisstudy.Asp27,Ser30,andSer272,highlighted in white with black background, have been mutated to cysteine for engineering a possible extra disulfide linkage between the N terminus and EC3 loop of C5aR. Middle and right panel, respectively, illustrate the effect of S30C, S272C, S30C/S272C, and D27C, S272C, D27C/S272C on C5a-stimulated C5aR signaling. Vector repre- sents the -galactosidase activity of the engineered yeast in the absence of both receptor and ligand. Each bar represents means S.D. of signaling activity for three independent transformants, and data are representative of at least two independent experiments.

Article Snippet: The emulsion was heated for 10 min at 50 °C, cooled on ice, and centrifuged for 5 min. 25 l of each supernatant was resolved on a NuPAGE Novex 4–12% BisTris gel, transferred to nitrocellulose membrane, and immunoblotted with a rabbit polyclonal anti-C5aR antibody raised against full-length endogenous C5aR (Novus Biologicals, Littleton, CO).

Techniques: Mutagenesis, Plasmid Preparation, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Third Extracellular Loop (EC3)-N Terminus Interaction Is Important for Seven-transmembrane Domain Receptor Function

doi: 10.1074/jbc.m110.129213

Figure Lengend Snippet: FIGURE 4. Signaling profile of wild type and mutant C5aR receptors, co- expressed with wild type and C27R C5a in yeast. The nonfunctional mutants, such as S272A and S272T, have not been tested with C27R C5a. Vector represents the -galactosidase activity of the engineered yeast in the absence of both receptor and ligand. Each bar represents means S.D. of signaling activity for three independent transformants, and data are repre- sentative of at least two independent experiments.

Article Snippet: The emulsion was heated for 10 min at 50 °C, cooled on ice, and centrifuged for 5 min. 25 l of each supernatant was resolved on a NuPAGE Novex 4–12% BisTris gel, transferred to nitrocellulose membrane, and immunoblotted with a rabbit polyclonal anti-C5aR antibody raised against full-length endogenous C5aR (Novus Biologicals, Littleton, CO).

Techniques: Mutagenesis, Plasmid Preparation, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Third Extracellular Loop (EC3)-N Terminus Interaction Is Important for Seven-transmembrane Domain Receptor Function

doi: 10.1074/jbc.m110.129213

Figure Lengend Snippet: FIGURE 5. Effect of point mutations on expression profile of C5aR in yeast as assessed by Western blot. 25 l of whole yeast lysates expressing the mutant or the wild type receptors were resolved on a 4–12% BisTris gel and detected as described under “Experimental Procedures.” The single and dou- ble asterisks, respectively, indicate the full-length and proteolytic fragments of the receptors. n/a, not applicable.

Article Snippet: The emulsion was heated for 10 min at 50 °C, cooled on ice, and centrifuged for 5 min. 25 l of each supernatant was resolved on a NuPAGE Novex 4–12% BisTris gel, transferred to nitrocellulose membrane, and immunoblotted with a rabbit polyclonal anti-C5aR antibody raised against full-length endogenous C5aR (Novus Biologicals, Littleton, CO).

Techniques: Expressing, Western Blot, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: Third Extracellular Loop (EC3)-N Terminus Interaction Is Important for Seven-transmembrane Domain Receptor Function

doi: 10.1074/jbc.m110.129213

Figure Lengend Snippet: FIGURE 6. Comparison of signaling profile of wild type, D282A, and S272A mutant C5aR receptors, co-expressed with C5a and metabolite C5a-des-Arg74 in yeast. C5a-des-Arg74 stimulates only 30% of the signal- ing in wild type C5aR compared with the signaling stimulated in response to C5a. D282A mutant displays reduced signaling in response to C5a consistent with other studies but displays near-maximal signaling in response to metab- olite C5a-des-Arg74. The nonfunctional mutant S272A neither responds to C5a nor to the metabolite C5a-des-Arg74. Vector represents the -galactosid- ase activity of the engineered yeast in the absence of both receptor and ligand. Each bar represents means S.D. of signaling activity for three inde- pendent transformants, and data are representative of at least two indepen- dent experiments.

Article Snippet: The emulsion was heated for 10 min at 50 °C, cooled on ice, and centrifuged for 5 min. 25 l of each supernatant was resolved on a NuPAGE Novex 4–12% BisTris gel, transferred to nitrocellulose membrane, and immunoblotted with a rabbit polyclonal anti-C5aR antibody raised against full-length endogenous C5aR (Novus Biologicals, Littleton, CO).

Techniques: Comparison, Mutagenesis, Plasmid Preparation, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Third Extracellular Loop (EC3)-N Terminus Interaction Is Important for Seven-transmembrane Domain Receptor Function

doi: 10.1074/jbc.m110.129213

Figure Lengend Snippet: FIGURE 8. Positional effect of N-terminal and EC3 cysteines on constitu- tive signaling profile of C5aR and their respective response to C5a, co- expressed in yeast. Left and right panels, respectively, illustrate the effect of S30C, S272C, S30C/S272C, and D27C, S272C, D27C/S272C on ligand-indepen- dent and -dependent signaling of constitutively active C5aR. The contrasting effect of S30C/S272C, D27C/S272C, and S272C on constitutive signaling of C5aR is noted in both panels. Vector represents the -galactosidase activity of the engineered yeast in the absence of both receptor and ligand. Each bar represents means S.D. of signaling activity for three independent transfor- mants, and data are representative of at least two independent experiments.

Article Snippet: The emulsion was heated for 10 min at 50 °C, cooled on ice, and centrifuged for 5 min. 25 l of each supernatant was resolved on a NuPAGE Novex 4–12% BisTris gel, transferred to nitrocellulose membrane, and immunoblotted with a rabbit polyclonal anti-C5aR antibody raised against full-length endogenous C5aR (Novus Biologicals, Littleton, CO).

Techniques: Plasmid Preparation, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Third Extracellular Loop (EC3)-N Terminus Interaction Is Important for Seven-transmembrane Domain Receptor Function

doi: 10.1074/jbc.m110.129213

Figure Lengend Snippet: FIGURE 9. Probing the side chain effect at Ser272 of EC3 on constitutive signaling of C5aR in yeast. The constitutive signaling profile of S272T, S272A, and S272C mutants in the presence and absence of S30C at the N terminus of C5aR. Maximal signaling is noted for all the single and double mutants in response to C5a. Vector represents the -galactosidase activity of the engineered yeast in the absence of both receptor and ligand. Each bar represents means S.D. of signaling activity for three independent transfor- mants, and data are representative of at least two independent experiments.

Article Snippet: The emulsion was heated for 10 min at 50 °C, cooled on ice, and centrifuged for 5 min. 25 l of each supernatant was resolved on a NuPAGE Novex 4–12% BisTris gel, transferred to nitrocellulose membrane, and immunoblotted with a rabbit polyclonal anti-C5aR antibody raised against full-length endogenous C5aR (Novus Biologicals, Littleton, CO).

Techniques: Plasmid Preparation, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Third Extracellular Loop (EC3)-N Terminus Interaction Is Important for Seven-transmembrane Domain Receptor Function

doi: 10.1074/jbc.m110.129213

Figure Lengend Snippet: FIGURE10.SignalingassaysinCOS-7cellsexpressingG16withwildtype or mutant receptors. Left and right panels, respectively, represent C5aR and CXCR4. The cells were stimulated with 10 nM C5a (C5aR) or 100 nM CXCL12 (CXCR4). IP3 accumulation values are normalized to the mock transfections, where each bar represents means S.D. of three independent experiments.

Article Snippet: The emulsion was heated for 10 min at 50 °C, cooled on ice, and centrifuged for 5 min. 25 l of each supernatant was resolved on a NuPAGE Novex 4–12% BisTris gel, transferred to nitrocellulose membrane, and immunoblotted with a rabbit polyclonal anti-C5aR antibody raised against full-length endogenous C5aR (Novus Biologicals, Littleton, CO).

Techniques: Mutagenesis, Transfection

Journal: Journal of Biological Chemistry

Article Title: Third Extracellular Loop (EC3)-N Terminus Interaction Is Important for Seven-transmembrane Domain Receptor Function

doi: 10.1074/jbc.m110.129213

Figure Lengend Snippet: FIGURE 11. Hypothetical molecular models of C5aR and illustration of conformational changes in EC3 due to disulfide linkage and receptor activation. Molecular models of wild type and engineered C5aR are pre- sented, respectively, in A and B. Asp27, Ser30, Asn119, and Ser272 that have been mutated for engineering the C5aR are highlighted in spheres. B, highlight of both the conserved and the engineered disulfide bond, respectively, between Cys109–Cys188 and Cys30–Cys272 in spheres, in con- text of other residues. Loop structures have been smoothed for clarity. C, structural alignment of EC3 of wild type (green loop with cyan helices, 1F88) and engineered rhodopsin (magenta loop with sand helices, Protein Data Bank code 2J4Y). Disulfide linkage between Asp282 of EC3 with Asn2 of the N terminus increases the EC3 loop length by straightening the TM7 by one helical loop. D, structural alignment of EC3 of rhodopsin (green loop with cyan helices, Protein Data Bank code 1F88) with EC3 of appar- ently active opsin (red loop with lime helices, Protein Data Bank code 3DQB). Major conformational changes noted in EC3 are associated with straightening of both TM6 and TM7 helices.

Article Snippet: The emulsion was heated for 10 min at 50 °C, cooled on ice, and centrifuged for 5 min. 25 l of each supernatant was resolved on a NuPAGE Novex 4–12% BisTris gel, transferred to nitrocellulose membrane, and immunoblotted with a rabbit polyclonal anti-C5aR antibody raised against full-length endogenous C5aR (Novus Biologicals, Littleton, CO).

Techniques: Activation Assay