Journal: Hepatobiliary Surgery and Nutrition

Article Title: EREG-secreting THBS1 + tissue monocytes are recruited by C5a to promote rapid liver regeneration in patients and mice during the ALPPS procedure

doi: 10.21037/hbsn-24-391

Figure Lengend Snippet: The potential role of C5a in the recruitment of THBS1 + tissue monocytes. (A) The results of a GO analysis of bulk FLR tissue. (B) Bubble plots of THBS1 + tissue monocyte receptor interactions (left) and hepatocyte ligand interactions (right) generated according to the CellphoneDB. The color is coded by the mean intensity of the interaction, and the size of the bubble is determined by the −log 10 P. (C) t-SNE plot showing clusters generated by scRNA-seq data. The color is coded by the expression level of C5a. (D) t-SNE plot showing MP clusters generated by scRNA-seq data, and a violin plot showing the C5aR1 expression in all Mo/Mφ populations. (E) GO and KEGG analyses of genes up-regulated in stage II hepatocytes as revealed by scRNA-seq. FLR, future liver remnant; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; Mo, monocytes; MP, Mo/Mφ population; Mφ, macrophages; scRNA-seq, single-cell RNA sequencing; t-SNE, t-distributed stochastic neighbor embedding.

Article Snippet: For C5a blockade, PMX-53, a specific C5aR1 antagonist, was used (HY-106178, MedChemExpress, Shanghai, China).

Techniques: Generated, Expressing, Single Cell, RNA Sequencing

Journal: Hepatobiliary Surgery and Nutrition

Article Title: EREG-secreting THBS1 + tissue monocytes are recruited by C5a to promote rapid liver regeneration in patients and mice during the ALPPS procedure

doi: 10.21037/hbsn-24-391

Figure Lengend Snippet: THBS1 + tissue monocytes were recruited through C5a. (A) Multiplexed immunofluorescence staining, showing the distribution of C5aR1 + monocytes (CD14 + C5aR1 + ). (B) Immunohistochemical staining of C5a at ALPPS stage I (upper left) and stage II (lower left). The H-score shows the up-regulation of C5a expression in hepatocytes from ALPPS stage I to stage II. *, P<0.05. (C) Liver samples from the murine ALPSS model on POD1 and POD5 (left). The boxplot represents comparisons of the FLR to body weight ratio between the control group and PMX-53 group on POD1 and POD5. (D) Immunohistochemical staining of Ki-67 for the control group and PMX-5 group on POD1 and POD5 (left). The boxplot shows comparisons of the ratios of Ki-67 + cells between the control and PMX-53 group on POD1 and POD5 (right). (E) Boxplot showing the EREG expression in the control and PMX-53 group on POD1 and POD5. (F) Schematic diagram of the ALPPS-induced liver regeneration. *, P<0.05 (actual P values were reported in results). ALPPS, associating liver partition and portal vein ligation for staged hepatectomy; EREG, epiregulin; IF, immunofluorescence; IHC, immunohistochemistry; FLR, future liver remnant; POD, post-operation day.

Article Snippet: For C5a blockade, PMX-53, a specific C5aR1 antagonist, was used (HY-106178, MedChemExpress, Shanghai, China).

Techniques: Immunofluorescence, Staining, Immunohistochemical staining, Expressing, Control, Ligation, Immunohistochemistry

Journal: Nature Communications

Article Title: Schwann cell C5aR1 co-opts inflammasome NLRP1 to sustain pain in a mouse model of endometriosis

doi: 10.1038/s41467-024-54486-6

Figure Lengend Snippet: Representative images of SOX10 and C5aR1 (#orb393229, rabbit polyclonal, Biorbyt Ltd, 1:100) co-expression in sciatic ( a ) and trigeminal ( b ) nerve tissue from C57BL/6 J female (B6) mice, ( n = 4 independent experiments) (Scale bar, 50 μm). (c) C5aR1 mRNA relative expression in primary mouse and human Schwann cells ( n = 3 independent experiments). d Schematic representation of AAV-(loxP-shRNA)- C5aR1 vector pre- and post-Cre switch. e Representative images and cumulative data (Rcoloc) of SOX10 and C5aR1 (#orb393229, rabbit polyclonal, Biorbyt Ltd, 1:100) co-expression in sciatic and trigeminal nerve tissue in Plp -AAV- C5aR1 and Control mice ( n = 4 independent experiments) (Scale bar, 50 μm). f Time-dependent periorbital (PMA), hind paw (HMA), and abdominal (AMA) mechanical allodynia in endometriotic (endo) or Sham Plp -AAV- C5aR1 and Control mice. ( n = 8 mice per group). g Representative images and cumulative data of F4/80 + cells in sciatic and trigeminal nerve tissue in endo or Sham Plp -AAV- C5aR1 and Control mice. ( n = 4 independent experiments) (Scale bar, 50 μm, dashed lines, perineurium ). h Representative images and cumulative data of transmigrated macrophages after stimulation of human and mouse Schwann cells with C5a or vehicle (Veh) and in the presence of DF2593A (DF25) or Veh ( n = 6 independent experiments). Data are mean ± s.e.m. c , Student’s t-test, f , 2-way, g, h 1-way ANOVA, Bonferroni correction; **** P < 0.0001 vs. Sham/Control, #### P < 0.0001 vs. Endo/Control. Source data are provided as a Source Data file.

Article Snippet: Representative images of SOX10 and C5aR1 (#orb393229, rabbit polyclonal, Biorbyt Ltd, 1:100) co-expression in sciatic ( a ) and trigeminal ( b ) nerve tissue from C57BL/6 J female (B6) mice, ( n = 4 independent experiments) (Scale bar, 50 μm). (c) C5aR1 mRNA relative expression in primary mouse and human Schwann cells ( n = 3 independent experiments). d Schematic representation of AAV-(loxP-shRNA)- C5aR1 vector pre- and post-Cre switch. e Representative images and cumulative data (Rcoloc) of SOX10 and C5aR1 (#orb393229, rabbit polyclonal, Biorbyt Ltd, 1:100) co-expression in sciatic and trigeminal nerve tissue in Plp -AAV- C5aR1 and Control mice ( n = 4 independent experiments) (Scale bar, 50 μm). f Time-dependent periorbital (PMA), hind paw (HMA), and abdominal (AMA) mechanical allodynia in endometriotic (endo) or Sham Plp -AAV- C5aR1 and Control mice. ( n = 8 mice per group). g Representative images and cumulative data of F4/80 + cells in sciatic and trigeminal nerve tissue in endo or Sham Plp -AAV- C5aR1 and Control mice. ( n = 4 independent experiments) (Scale bar, 50 μm, dashed lines, perineurium ). h Representative images and cumulative data of transmigrated macrophages after stimulation of human and mouse Schwann cells with C5a or vehicle (Veh) and in the presence of DF2593A (DF25) or Veh ( n = 6 independent experiments).

Techniques: Expressing, shRNA, Plasmid Preparation, Control

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: 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: 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