Journal: Scientific Reports

Article Title: Cardiac Depression in Pigs after Multiple Trauma – Characterization of Posttraumatic Structural and Functional Alterations

doi: 10.1038/s41598-017-18088-1

Figure Lengend Snippet: Local inflammation in cardiac left ventricular tissue 72 h after multiple trauma or sham procedure. ( A ) Elevation of proinflammatory cardiodepressive cytokine IL-1β concentration in left ventricular homogenates after multiple trauma in both treatment groups DCO (grey bar) and ETC (black bar) compared to animals after sham procedure (white bar) assessed by ELISA. ( B ) Elevation of proinflammatory cardiodepressive cytokine IL-6 concentration in left ventricular homogenates after multiple trauma in ETC (black bar) treatment group compared to animals after sham procedure (white bar) assessed by ELISA. ( C ) Representative western blot of C5aR1 in left ventricular homogenates in pigs after trauma and DCO (grey bar) or ETC (black bar) treatment and in sham treated animals (white bar), pixel densitry was assessed for quantification. Representitive C5aR1 staining of left ventricle tissue after trauma and DCO (middle) or ETC (right) treatment and in sham (left) treated animals ( D ). ( E ) Changes in density of C5aR1 staining in left ventricular tissue after trauma and DCO (grey bar) or ETC (black bar) treatment and in sham treated animals (white bar). ( F ) Changes in C5aR2 protein expression in left ventricular tissue after trauma and DCO (grey bar) or ETC (black bar) treatment and in sham treated animals (white bar). For each bar n = 6 samples, *p < 0.05.

Article Snippet: For C5aR1 staining polyclonal anti-CD88/C5aR1 (Acris Antibodies, Herford, Germany) was used as primary antibody.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Staining, Expressing

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: ( A ) Gene Set Enrichment Analysis (GSEA) identified GLUT1 and C5aR1 as two top hits related to selective serotonin reuptake inhibitor (SSRI)-induced gene changes. ( B ) GSEA of hepatocellular carcinoma (HCC) RNA-seq data (TCGA cohort) with the SSRI-related gene signature. Sample grouping was made based on the median expression of C5aR1. ( C ) Representative immunohistochemical images showed the expression pattern and cellular distribution of C5aR1 in human HCC tissues. Scale bar, 50 μm. ( D ) Single-cell RNA sequencing analysis showed the expression pattern of C5aR1 with the immune microenvironment of HCC. ( E ) Co-immunofluorescence of C5aR1 (green) with CD163 (red) in HCC samples. Scale bar, 10 μm. ( F ) The drug affinity responsive target stability (DARTS) assay and immunoblot analysis showed C5aR1 protein stability against 5 μg/ml pronase in the presence and absence of 100 μM citalopram treatment. ( G ) The DARTS assay and immunoblot analysis showed C5aR1 protein stability against 5 μg/ml pronase in the presence of different concentrations of citalopram treatment. ( H ) The overall conformation of citalopram binding to C5aR1. ( I ) Representative models of citalopram in pose-1 (left), pose-2 (middle), and allosteric site (right). Several polar interactions were indicated by black dashed lines. ( J ) HEK293T cells were transfected with either WT or mutant C5aR1 expression plasmids for 48 hr, followed by DARTS assay with immunoblotting analysis of C5aR1 protein levels. In all panels, *p < 0.05, **p < 0.01. Values as mean ± SD and compared by the Student’s t test ( F ) or one-way analysis of variance (ANOVA) multiple comparisons with Tukey’s method among groups ( G, J ). Figure 2—source data 1. Original western blots for , indicating the relevant bands and treatments. Figure 2—source data 2. Original files for western blot analysis displayed in .

Article Snippet: The following antibodies were used: C5aR1 (1:50, Proteintech, 10375-1-AP) and CD163 (1:200, Abcam, ab182422).

Techniques: RNA Sequencing, Expressing, Immunohistochemical staining, Single Cell, Immunofluorescence, Western Blot, Binding Assay, Transfection, Mutagenesis

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: ( A ) Uniform Manifold Approximation and Projection (UMAP) of SMART-seq2-based single CD45 + cells. The tSNE (by cluster) was acquired from http://cancer-pku.cn:3838/HCC/ . ( B ) The UMAP showing C5AR1 expression in HCC immune cell clusters. ( C ) Violin plot showing C5AR1 expression in different immune cell clusters.

Article Snippet: The following antibodies were used: C5aR1 (1:50, Proteintech, 10375-1-AP) and CD163 (1:200, Abcam, ab182422).

Techniques: Expressing

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: ( A ) The drug affinity responsive target stability (DARTS) assay and immunoblot analysis showed C5aR1 protein stability against 5 μg/ml pronase in the presence of different concentrations of selective serotonin reuptake inhibitors (SSRIs) treatment (0, 1, 10, 50, and 100 μM). ( B ) For C5aR1, the predicted binding energy distribution of the clusters with poses more than 50. ( C ) Sequencing analysis showed the successful generation of six C5aR1 mutants. ( D ) The best-scored complex models of C5aR1 with other four different SSRIs. ( E ) HEK293T cells were transfected with either WT or mutant C5aR1 (D282A) expression plasmids for 48 hr, followed by DARTS assay with immunoblotting analysis of C5aR1 protein levels. In all panels, *p < 0.05, **p < 0.01. Values are presented as mean ± SD and compared by one-way analysis of variance (ANOVA) multiple comparisons with Tukey’s method among groups ( E ). Figure 2—figure supplement 2—source data 1. Original western blots for , indicating the relevant bands and treatments. Figure 2—figure supplement 2—source data 2. Original files for western blot analysis displayed in .

Article Snippet: The following antibodies were used: C5aR1 (1:50, Proteintech, 10375-1-AP) and CD163 (1:200, Abcam, ab182422).

Techniques: Western Blot, Binding Assay, Sequencing, Transfection, Mutagenesis, Expressing

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: ( A ) Western blotting showed the knockdown efficiency of GLUT1 in mouse Hepa1-6 cells. ( B ) GLUT1 KD Hepa1-6 cells were subcutaneously injected into the Rag1 −/− or immunocompetent C57BL/6 mice, and mice were treated with 5 mg/kg citalopram when bore visible tumors; 3 weeks later, tumor burden was examined ( n = 6–7 per group). ( C ) The growth kinetics of GLUT1 KD Hepa1-6 tumors in C5ar1 +/− and C5ar1 −/− C57BL/6 host ( n = 7). ( D ) Immunofluorescence analysis of C5a deposition in GLUT1 KD Hepa1-6 tumors from C5ar1 +/− and C5ar1 −/− C57BL/6 host. Scale bar, 50 μm. ( E ) Experimental design of bone marrow transfer experiments. ( F, G, I ) GLUT1 KD Hepa1-6 cells were subcutaneously implanted into syngeneic recipient (r) mice that had been reconstituted with bone marrow cells from either C5ar1 +/− or C5ar1 −/− donor mice. The therapeutic effect of citalopram ( F ), C5a deposition ( G ), and macrophage phagocytosis ( I ) in this model was analyzed. Scale bar, 50 μm. ( H ) The phagocytic capacity of macrophages isolated from GLUT1 KD Hepa1-6 tumors in C5ar1 +/− and C5ar1 −/− C57BL/6 host. Flow cytometry showed the infiltration of CD45 + CD11b + F4/80 + macrophages ( J ), CD206 + TAMs and CD11b + TAMs ( K ), tumor-infiltrating lymphocytes ( L ) in tumor tissues from orthotopic xenograft model, which was generated in immunocompetent C57BL/6 mice with Hepa1-6 cells ( n = 5 per group). ( M, N ) Measurement of CD8 + T cell function in tumor tissues from the groups mentioned in C and F . ( O ) The growth kinetics of GLUT1 KD Hepa1-6 tumors in C5ar1 +/− and C5ar1 −/− C57BL/6 host upon CD8 + T cell depletion ( n = 7). ( P ) Correlation analysis of C5aR1 expression and immune checkpoint molecules, gene signatures of TAMs, exhausted T cells, and effector Tregs in the TCGA cohort ( n = 371). In all panels, *p < 0.05, **p < 0.01, ***p < 0.001; ns, non-significant. Values are presented as mean ± SD and compared by two-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons ( B, C, F, O ), Student’s t test ( H–M ), one-way ANOVA multiple comparisons with Tukey’s method ( B, N ), and the Spearman’s rank correlation methods ( P ). Figure 3—source data 1. Original western blots for , indicating the relevant bands. Figure 3—source data 2. Original files for western blot analysis displayed in .

Article Snippet: The following antibodies were used: C5aR1 (1:50, Proteintech, 10375-1-AP) and CD163 (1:200, Abcam, ab182422).

Techniques: Western Blot, Knockdown, Injection, Immunofluorescence, Isolation, Flow Cytometry, Generated, Cell Function Assay, Expressing

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: ( A ) Gene Set Enrichment Analysis (GSEA) plot of phagocytosis pathway in macrophages derived from C5ar1 −/− mice and C5ar1 +/− mice. ( B ) Western blotting and immunofluorescence analysis showed C5aR1 protein levels in Cas9-sgControl, -sg C5ar1 THP-1 subclones. ( C ) Effects of C5aR1 deficiency on the macrophage phagocytosis of HCC-LM3 in the presence or absence of C5a stimulation. ( D ) Effects of different selective serotonin reuptake inhibitors (SSRIs) on the macrophage phagocytosis of HCC-LM3 in the presence of C5a stimulation. ( E ) Reconstituted expression of WT and D282A mutant C5aR1 in C5aR1 KO THP-1 cells. ( F ) The effects of citalopram on macrophage phagocytosis in the absence of C5aR1 with reconstituted expression of C5aR1 WT or C5aR1 D282A . In all panels, *p < 0.05, **p < 0.01, ***p < 0.001. Values are presented as mean ± SD and compared by one-way analysis of variance (ANOVA) multiple comparisons with Tukey’s method among groups. Data are representative of three independent experiments ( C, D, F ). Figure 3—figure supplement 2—source data 1. Original western blots for , indicating the relevant bands. Figure 3—figure supplement 2—source data 2. Original files for western blot analysis displayed in .

Article Snippet: The following antibodies were used: C5aR1 (1:50, Proteintech, 10375-1-AP) and CD163 (1:200, Abcam, ab182422).

Techniques: Derivative Assay, Western Blot, Immunofluorescence, Expressing, Mutagenesis

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: ( A ) Conformations of orthosteric binding sites in human (light blue) and mouse (orange) C5aR1. The conformation of human C5aR1 was obtained from the crystal structure (PDB id: 6c1q). The structure of mouse C5aR1 was predicted using the ColabFold (AlphaFold2) software. ( B ) The predicted binding modes of citalopram to human (light blue) and mouse (orange) C5aR1. The conformations of citalopram were shown in pink (binding mode 1) or deep green (binding mode 2) sticks. For mouse C5aR1, green sticks indicate residues set to flexible in the molecular docking process.

Article Snippet: The following antibodies were used: C5aR1 (1:50, Proteintech, 10375-1-AP) and CD163 (1:200, Abcam, ab182422).

Techniques: Binding Assay, Software

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: Model depicting the molecular mechanism by which citalopram inhibits the Warburg effect and promotes an anti-tumor response in hepatocellular carcinoma (HCC). In the primary HCC microenvironment (left panel), C5aR1-expressing tumor-associated macrophages (TAMs) exhibit reduced phagocytic capacity and an anti-inflammatory state, which correlates with diminished CD8 + T cell anti-tumor immunity and HCC progression. Upon treatment with citalopram (right panel), the drug not only inhibits the glycolytic metabolism of cancer cells by targeting GLUT1 but also acts on C5aR1 expressed by TAMs, thereby enhancing macrophage-driven anti-tumor immunity. Additionally, citalopram induces a systemic immunostimulatory effect on CD8 + T cell functions through yet-to-be-identified serotonergic mechanisms. The dotted line indicates a causal relationship that has not been fully established through direct evidence.

Article Snippet: The following antibodies were used: C5aR1 (1:50, Proteintech, 10375-1-AP) and CD163 (1:200, Abcam, ab182422).

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: Complement C5a exacerbates acute lung injury induced through autophagy-mediated alveolar macrophage apoptosis

doi: 10.1038/cddis.2014.274

Figure Lengend Snippet: C5a production induces alveolar macrophage activation in acute lung injury. ( a ) Eight-week-old C57BL/6 male mice or C5aR −/− male mice (six mice per group) were subjected to intestinal ischemia/reperfusion and BAL fluid was collected 45 min later. TNF- α , IL-6, and MCP-1 were measured by ELISA. Data are from three independent experiments. ( b ) Alveolar macrophages prepared as described in Materials and Methods were stained with F4/80, CD11b, or CD80, and subjected to FACS analysis. ( c ) Alveolar macrophages were cultured, and supernatants were collected after 12 h. TNF- α , IL-6, and MCP-1 were measured by ELISA. Data are the mean±S.D. of three independent experiments. * P <0.05

Article Snippet: The anti-mouse C5aR antibody was obtained from OriGene (Rockville, MD, USA).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Staining, Cell Culture

Journal: Cell Death & Disease

Article Title: Complement C5a exacerbates acute lung injury induced through autophagy-mediated alveolar macrophage apoptosis

doi: 10.1038/cddis.2014.274

Figure Lengend Snippet: C5a induces alveolar macrophage autophagy through Bcl-2 degradation. ( a ) Alveolar macrophages were isolated from normal or ALI mice and lysed. Lysates were analyzed by immunoblotting with LC3 antibody. ( b ) Alveolar macrophages were isolated from ARDS patients and volunteers and lysed. Lysates were analyzed by immunoblotting with LC3 antibody and C5aR antibody. ( c ) Normal mice, ALI mice, and ALI mice pretreated intravenously with C5a antibody or C5aR antibodies were killed and frozen lung tissue sections were obtained and stained with Alexa 488-F4/80, Alexa-594-LC3, and nucleus counterstained with DAPI. ( d ) Alveolar macrophages were isolated from mice and lysates were analyzed by immunoblotting with Beclin 1 antibody. ( e ) Alveolar macrophages were isolated from mice and lysates were analyzed by immunoblotting with Bcl-2 antibody. Quantitative data are the mean±S.D. of three independent experiments. * P <0.05

Article Snippet: The anti-mouse C5aR antibody was obtained from OriGene (Rockville, MD, USA).

Techniques: Isolation, Western Blot, Staining

Journal: The Journal of Immunology Author Choice

Article Title: Endoplasmic Reticulum Stress of Neutrophils Is Required for Ischemia/Reperfusion–Induced Acute Lung Injury

doi: 10.4049/jimmunol.1500073

Figure Lengend Snippet: C5a induces ER stress of neutrophil. (A) Eight-week-old C57BL/6 male mice (six mice per group) were subjected to intestinal IR. BALF was collected 60 min after the procedures. C5a was measured using ELISA. Data are from three independent experiments. (B) Neutrophils prepared from mice peripheral blood or murine myeloid cell line 32Dcl3 were treated with C5a for 4 h and then lysed. Lysates were analyzed by immunoblotting with indicated Abs. Images shown are representatives of three experiments. (C) Neutrophils prepared from mice peripheral blood were treated with C5a alone or combined with pertussis toxin (PTX) for 4 h and then lysed. Lysates were analyzed by immunoblotting with indicated Abs. Images shown are representatives of three experiments. (D) Neutrophils prepared from mouse peripheral blood were treated with fMLF (200 nM) or PMA (100 nM) for 60 min and then lysed. Lysates were analyzed by immunoblotting with indicated Abs. Images shown are representatives of three experiments. (E) Supernatants from cultured neutrophils described as in (B) were collected, and MPO was measured using ELISA. (F) Murine myeloid cells, 32Dcl3, were transiently transfected with siXBP1 overnight, followed by 4 h of C5a treatment or 4 h of starvation and then lysed. Lysates were analyzed by immunoblotting with indicated Abs. Images shown are representatives of three experiments. Quantification is expressed as mean ± SD of three independent experiments. *p < 0.05.

Article Snippet: The anti-mouse complement 5a (C5a) receptor (C5aR) Ab was obtained from OriGene (Rockville, MD).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Cell Culture, Transfection

Journal: The Journal of Immunology Author Choice

Article Title: Endoplasmic Reticulum Stress of Neutrophils Is Required for Ischemia/Reperfusion–Induced Acute Lung Injury

doi: 10.4049/jimmunol.1500073

Figure Lengend Snippet: C5a interacting with C5aR induces ER stress in neutrophils. (A) Neutrophils prepared from mouse peripheral blood or murine myeloid cell line 32Dcl3 were lysed for immunoblotting with C5aR Ab. Images shown are representative of three experiments. Band intensities were determined with ImageJ. (B) Murine myeloid cells (32Dcl3) were transiently transfected with siC5aR overnight and then lysed. Lysates were analyzed by immunoblotting with C5aR Ab. Images shown are representatives of three experiments. (C) Murine myeloid cells (32Dcl3) were transiently transfected with siC5aR overnight, followed by treatment with C5a and/or C5a neutralizing Ab for 4 h and then lysed. Lysates were analyzed by immunoblotting with indicated Abs. Images shown are representatives of three experiments. (D) Supernatants from cultured neutrophils described as in (C) were collected, and MPO was measured using ELISA. Data are expressed as mean ± SD of three independent experiments. *p < 0.05.

Article Snippet: The anti-mouse complement 5a (C5a) receptor (C5aR) Ab was obtained from OriGene (Rockville, MD).

Techniques: Western Blot, Transfection, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Immunology Author Choice

Article Title: Endoplasmic Reticulum Stress of Neutrophils Is Required for Ischemia/Reperfusion–Induced Acute Lung Injury

doi: 10.4049/jimmunol.1500073

Figure Lengend Snippet: Inhibition of ER stress in neutrophils decreases ALI in vivo. (A) xbpf/f MRP8-cre mice (KO) and XBP1f/f mice were used to establish IR-induced ALI model. Lungs samples were fixed and embedded in paraffin. Tissue blocks were sectioned at 5 μm and stained with H&E. Morphology was examined using light microscopy. Pathological scores were given by an experienced pathologist. (B) Sections were stained with Ly6G (1A8) Ab. (C) Neutrophils were prepared from the BALF of normal or ALI xbpf/f MRP8-cre mice and XBP1fl/fl mice, and they were subjected to immunofluorescence staining with indicated Abs. Images shown are representatives of three experiments. (D) Neutrophils were prepared from the BALF of normal or ALI xbpf/f MRP8-cre mice and XBP1fl/fl mice and then lysed. Lysates were analyzed by immunoblotting with indicated Abs. Images shown are representatives of three experiments. (E) BALF was collected from xbpf/f MRP8-cre mice (KO/N) and XBP1fl/fl mice (fl/fl). TNF-α, IL-6, C5a, and MPO were measured using ELISA. Quantification is expressed as mean ± SD of three independent experiments. Scale bars, 50 μm. *p < 0.05.

Article Snippet: The anti-mouse complement 5a (C5a) receptor (C5aR) Ab was obtained from OriGene (Rockville, MD).

Techniques: Inhibition, In Vivo, Staining, Light Microscopy, Immunofluorescence, Western Blot, Enzyme-linked Immunosorbent 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 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

Journal: Scientific Reports

Article Title: Therapeutic effect of pH responsive Magainin II modified azithromycin plus curcumin micelles in different depth models of MRSA infection

doi: 10.1038/s41598-025-92384-z

Figure Lengend Snippet: Antibacterial effect of different micellar preparations on mice with pneumonia. ( A ) Real-time imaging observation of lungs treated with varying formulations in vivo. a, for Blank-Ms. B, for free DiR. c, for DiR-Ms. d, for MagII-DiR-Ms. ( B ) Representative images of lung tissue homogenate plate spreading. ( C ) Representative images of pathological changes in lungs by HE staining. ( D – G ) Expression of C5aR1, C5b-9, C3c and NF-κB p65 detected by immunohistochemistry in lungs. ( H – J ) Expression of inflammatory factors in BALF after treatment with different formulations. ( K – M ) Number of inflammatory cells in BALF after treatment with different micelles. Scale bar = 50 μm. Data are presented as mean ± SD ( n = 6), * P < 0.05, ** P < 0.01, **** P < 0.005.

Article Snippet: C5aR1 and NF-κB p65 antibodies were purchased from Boster Co., Ltd. (Wuhan, China).

Techniques: Imaging, In Vivo, Staining, Expressing, Immunohistochemistry