Journal: Computational and Structural Biotechnology Journal

Article Title: The role of microglia and complement C5/C5a in the pathogenesis of rhegmatogenous retinal detachment with choroidal detachment

doi: 10.1016/j.csbj.2025.08.019

Figure Lengend Snippet: scRNA-seq Analysis of Complement C5 Components and Intercellular Communication in Retinal Detachment. (A) UMAP feature plots (left) show C5, C5AR1, and C5AR2 expression across retinal cell clusters in RRD and RRDCD, with color intensity indicating expression levels. Violin plots (right) compare cell type-specific expression distributions between conditions. (B) CellPhoneDB-derived dot plot displays ligand-receptor interactions, with bubble size reflecting interaction significance (-log₁₀ p-value) and color denoting mean expression. Comparisons include C5-C5AR1, C3-C5AR2, TNF-TNFRSF1A/B, and VEGF (VEGFA-FLT1, VEGFB-NRP1) pathways between RRD and RRDCD.

Article Snippet: Experimental groups were treated with complement C5 (HY-P7695; MedChemExpress) at concentrations of 0.5 μg/mL, 1.0 μg/mL, and 2.5 μg/mL, while the control group received no complement C5.

Techniques: Expressing, Derivative Assay

Journal: Computational and Structural Biotechnology Journal

Article Title: The role of microglia and complement C5/C5a in the pathogenesis of rhegmatogenous retinal detachment with choroidal detachment

doi: 10.1016/j.csbj.2025.08.019

Figure Lengend Snippet: Characterization of complement C5 effects on cellular responses and molecular markers. (A) MTT assay assessing the viability/metabolic activity of RF/6 A choroidal vascular endothelial cells after 24-hour co-culture with primary mouse retinal microglia that were pre-treated with varying concentrations of complement C5 (0, 0.5, 1.0, 2.5 µg/mL)， with results expressed as ODsample/ODblank ratios. (B) Apoptotic effects of C5-treated (1 μg/mL, 24 h) microglia on RF/6 A choroidal vascular endothelial cells, as evidenced by TUNEL staining (red) and quantification of TUNEL-positive area. (C) C5 exposure (1 μg/mL, 24 h) altered microglial cell viability (MTT assay, Student's t -test) and morphology (brightfield images). (D) ZO-1 immunofluorescence (red) in ARPE-19 cells co-cultured with C5-treated retinal microglia (1 μg/mL, 24 h) showed tight junction integrity (nuclei: DAPI, blue). (E) CD86 expression (red) increased in retinal primary mouse microglia following C5 treatment (1 μg/mL, 24 h). (F) Western blot confirmed elevated CD86 protein levels in C5-treated microglia (1 μg/mL, 24 h). (G) ELISA detected significantly higher TNF-α and IL-1β secretion in C5-treated microglia (1 μg/mL, 24 h). Data represent mean ± SD of ≥ 3 independent experiments.

Article Snippet: Experimental groups were treated with complement C5 (HY-P7695; MedChemExpress) at concentrations of 0.5 μg/mL, 1.0 μg/mL, and 2.5 μg/mL, while the control group received no complement C5.

Techniques: MTT Assay, Activity Assay, Co-Culture Assay, TUNEL Assay, Staining, Immunofluorescence, Cell Culture, Expressing, Western Blot, Enzyme-linked Immunosorbent 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: Fig. 8. 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

Journal: Molecular Therapy

Article Title: A Novel C5a-neutralizing Mirror-image ( l -)Aptamer Prevents Organ Failure and Improves Survival in Experimental Sepsis

doi: 10.1038/mt.2013.178

Figure Lengend Snippet: NOX-D20 binds to C5 but does not inhibit complement-mediated hemolysis. SPR measurement of NOX-D20 binding to human ( a ) C5a, ( b ) C5a(desArg), and ( c ) C5. Kinetic rate constants k a and k d are shown as mean ± SEM. Data are representative for at least three individual measurements. ( d ) Human serum pretreated with NOX-D20 (black squares) or C5-binding aptamer C5C6 (black triangles) was incubated with opsonized sheep erythrocytes. Hemolysis was quantified by photometric measurement of hemoglobin in the supernatant at 405 nm. Normalized data representative for three independent experiments is shown. RU, response units.

Article Snippet: Recombinant human and mouse C5a was from R&D Systems (Wiesbaden, Germany), recombinant human C5a(desArg) from Hycult Biotech (Beutelsbach, Germany), and human C5 purified from serum from Sigma-Aldrich (Taufkirchen, Germany).

Techniques: Binding Assay, Incubation

Journal: Nature immunology

Article Title: Absent C3a and C5a receptor signaling into CD4 + T cells enables auto-inductive TGF-β1 signaling and induction of Foxp3 + T regulatory cells

doi: 10.1038/ni.2499

Figure Lengend Snippet: All inductions were performed for 3 days with 1 ×10 6 CD62L hi Foxp3 − CD25 − CD4 + T cells, rhIL-2 (5 ng/ml), and anti-CD3 + CD28 beads (per manufacturer’s instructions), experiments were repeated two times. ( a ) WT, C3ar1 −/− , C5ar1 −/− , or C3ar1 −/− C5ar1 −/− Foxp3 − cells were activated and assayed for percent Foxp3 + CD25 + by flow cytometry (*P<0.001, n=5). (b) Following iT reg induction (as in a ), flow sorted WT, C3ar1 −/− , C5ar1 −/− , or C3ar1 −/− C5ar1 −/− Foxp3 + CD25 + cells were incubated with 10 6 CellTracker Red™ labeled CD25 − Foxp3 − CD4 + WT cells in differing T eff /iT reg ratios + anti-CD3 (5 μg/ml) and 2.5×10 5 CD11c + DCs. Relative suppressive capacity was determined by percent Red-labeled dividers (n=5). (c) WT Foxp3 − CD4 + T cells were induced as in (a) in the absence or presence of anti-C3a (10 μg/ml), anti-C5a (10 μg/ml), or both or in the absence and presence of the antagonists C3aR-A (10 nM), C5aR-A (10 nM), or both. CD4 + T cells then were assayed for Foxp3 expression by flow cytometry (*P<0.001, n=6). (d) Following iT reg induction (as in a ), sorted WT and C3ar1 −/− C5ar1 −/− Foxp3 + CD25 + cells were washed and recultured for 24 hr in the absence of further stimulation. Culture supernatants were assayed for TGF-β, IL-6, and IL-10 by ELISA (*P<0.001, n=6). (e) C3ar1 −/− C5ar1 −/− iT regs were induced (as in a) in the absence and presence of anti-TGF-β mAb (5 μg/ml), TGF-βR1 inhibitor (10 nM), or Smad3 inhibitor (5 μM). Foxp3 + CD25 + T reg percentages were quantified by flow cytometry.

Article Snippet: Anti-Human C3a (20C-CR6032RP) was purchased from Fitzgerald and anti-human C5a (AF2037) was purchased from R&D Systems.

Techniques: Flow Cytometry, Incubation, Labeling, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Nature immunology

Article Title: Absent C3a and C5a receptor signaling into CD4 + T cells enables auto-inductive TGF-β1 signaling and induction of Foxp3 + T regulatory cells

doi: 10.1038/ni.2499

Figure Lengend Snippet: iT reg induction and activation experiments were performed as in with 2.5×10 5 CD11c + DCs and anti-CD3 mAb (5 μg/ml) instead of anti-CD3+CD28 beads, experiments were repeated two times. (a) Sorted WT and C3ar1 −/− C5ar1 −/− Foxp3 − CD4 + T cells were incubated with WT DCs ± TGF-β1 (5 ng/ml) and assayed for CD25 and Foxp3 expression by flow cytometry (*P<0.001, n=10). (b) (Left) WT Foxp3 − CD4 + T cells were incubated with C3 −/− C5 −/− DCs without TGF-β1 ± C3a/C5a (100 ng/ml) and Foxp3 + CD25 + cells quantified by flow cytometry. (Right) WT Foxp3 − CD4 + T cells were incubated in the presence of TGF-β1 (5 ng/ml) ± C3a/C5a (100 ng/ml) and percent Foxp3 + CD25 + cells quantified (*P<0.001, n=7). (c) Sorted WT or C3ar1 −/− C5ar1 −/− Foxp3 − CD4 + T cells were incubated with C3ar1 −/− C5ar1 −/− DCs in the absence or presence of C5a (100 ng/ml) after which percent Foxp3 + CD25 + cells was quantified. The % Foxp3 + cells with DKO or WT T cells did not significantly differ (P=0.54). (d) Sorted C3 −/− C5 −/− or C3ar1 −/− C5ar1 −/− Foxp3 − CD4 + T cells were incubated with Daf1 −/− DCs ± anti-C3a and anti-C5a mAbs after which percent Foxp3 + CD25 + cells was quantified (*P<0.001; n=6). (e) Sorted WT Foxp3 − or C3ar1 −/− C5ar1 −/− Foxp3 − CD4 + T cells were incubated for 24 hr either in the absence of DCs or in the presence of WT DCs or C3ar1 −/− C5ar1 −/− DCs. Supernatants then were assayed for TGF-β1 and IL-6 by ELISA (*P<0.001, n=6). (f) C3ar1 −/− C5ar1 −/− Foxp3 − cells were incubated for 3 days with anti-CD3 (5 μg/ml), IL-2 (5 ng/ml), and WT DCs ± anti-TGF-β mAb (5 μg/ml), TGF-βR1 inhibitor (10 nM), or Smad3 inhibitor (5 μM). Following the 3 day induction, Foxp3 + CD25 + T reg percentages were determined by flow cytometry.

Article Snippet: Anti-Human C3a (20C-CR6032RP) was purchased from Fitzgerald and anti-human C5a (AF2037) was purchased from R&D Systems.

Techniques: Activation Assay, Incubation, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Nature immunology

Article Title: Absent C3a and C5a receptor signaling into CD4 + T cells enables auto-inductive TGF-β1 signaling and induction of Foxp3 + T regulatory cells

doi: 10.1038/ni.2499

Figure Lengend Snippet: Experiments were repeated two times. (a) WT CD4 + T cells were incubated for 30 min with anti-CD3+CD28 beads after which they were incubated for 10 min with Forskolin (30 μM) ± C3a (100 nM), C5a (100 nM), or both. Levels of cAMP activity were determined by cAMP-Glo assay (*P<0.001). (b) WT, C3ar1 −/− , C5ar1 −/− , and C3ar1 −/− C5ar1 −/− CD4 + T cells were stimulated with anti-CD3 mAb for 30 min after which PKA activity was quantified by PepTag assay. (c) iT regs generated from sorted WT Foxp3 − CD4 + T cells plus TGF-β1, iT regs derived from C3ar1 −/− C5ar1 −/− Foxp3 − CD4 + T cells, and conventional Foxp3 − CD4 + T cells were stimulated with anti-CD3+CD28 beads for 15 min and assayed for S 473 and T 308 p-AKT by Luminex assay (n=5). (d and e) WT or C3ar1 −/− C5ar1 −/− CD4 + T cells were incubated for 15 min ± anti-CD3+CD28 beads. Cells were extracted in phospho-lysis buffer and extracts assayed for (d) phospho-rbS6 and (e) phospho-Smad2 by immunoblotting. (f) iT regs induced from WT Foxp3 − CD4 + T cells plus TGF-β1 or from C3ar1 −/− C5ar1 −/− Foxp3 − CD4 + T cells were assayed for phospho-STAT3 and phospho-STAT5 by flow cytometry (representative plots of n=6).

Article Snippet: Anti-Human C3a (20C-CR6032RP) was purchased from Fitzgerald and anti-human C5a (AF2037) was purchased from R&D Systems.

Techniques: Incubation, Activity Assay, Glo Assay, Generated, Derivative Assay, Luminex, Lysis, Western Blot, Flow Cytometry

Journal: Nature immunology

Article Title: Absent C3a and C5a receptor signaling into CD4 + T cells enables auto-inductive TGF-β1 signaling and induction of Foxp3 + T regulatory cells

doi: 10.1038/ni.2499

Figure Lengend Snippet: Experiments were repeated two times. (a) Sorted WT Foxp3 − cells were incubated for 1 hr with anti-CD3+CD28 beads (as in ) in the absence or presence of TGF-β1 (5 ng/ml) and then assayed for complement mRNA transcripts by qPCR (*P<0.001, n=5). (b) Sorted WT Foxp3 − CD4 + T cells were incubated for 1 hr with anti-CD3+CD28 beads plus TGF-β1 (5 ng/ml) alone, TGF-β1 (5 ng/ml) plus IL-6 (5 ng/ml), or IL-6 (5 ng/ml) alone, after which the cells were assayed for C3 mRNA expression by qPCR (*P<0.001, **P<0.02, n=5). (c and d) Sorted WT Foxp3 − CD4 + T cells were incubated for 48 hr with anti-CD3+CD28 beads plus TGF-β1 alone, TGF-β1+IL-6, or IL-6 alone as in (b) . Culture supernatants were assayed for (c) C3a and C5a generation by ELISAs, and (d) C3aR and C5aR surface expression by flow cytometry (P<0.01 for TGF-β1 alone vs TGF-β1+IL-6 or IL-6 alone, Mean Fluorescence Intensity (MFI) values; n=5). (e) Sorted WT Foxp3 − cells were activated for 48 hr with anti-CD3+CD28 beads ± C5a (100 ng/ml) or C3aR-A/C5aR-A (10 nM), after which culture supernatants were assayed for TGF-β1 or IL-6 by ELISA (P<0.05; n=6). (f) Sorted WT Foxp3 − CD4 + T cells were incubated with WT DCs or with C3 −/− C5 −/− DCs in the absence of TGF-β1 ( C3 −/− C5 −/− iT regs ). DCs (left side) were assayed for C5aR and C5L2 expression by gating on CD11c + cells. Responder T cells (right side) were assayed for C5aR and C5L2 expression by gating on Foxp3 − cells in the case of WT CD4 + T cells prepared with WT DCs and on Foxp3 + cells in the case of WT CD4 + prepared with C3 −/− C5 −/− DCs. (*P < 0.01; n=5). (g) Sorted WT or C5L2 −/− (encoded by Gpr77) Foxp3 − CD4 + T cells were incubated with WT DCs (Left) and sorted WT Foxp3 − CD4 + T cells were incubated with WT or C5L2 −/− DCs (right) both in the presence of anti-CD3 and TGF-β1 (5 ng/ml). Percent Foxp3 + CD25 + cells were assayed by flow cytometry. (*P<0.001, n=6). (h) WT Foxp3 − CD4 + T cells were incubated for 3 days with WT DCs plus TGF-β1 (5 ng/ml) after which Foxp3 + and Foxp3 − cells were sorted. Foxp3 − CD4 + T cells or Foxp3 + CD4 + iT regs were incubated for 5 min with anti-CD3+CD28 beads + biotin-labeled C5a, the cells chilled to 4°C, and plasma membrane fractions were isolated by ultracentrifugation. Following C5aR and C5L2 IP, bound proteins were eluted with alkaline solution, and equal amounts of protein (determined by A 280 ) were loaded onto SDS PAGE gels. Following electrophoresis, gels were blotted for biotinylated-C5a by adding streptavidin-HRP followed by ECL reagent.

Article Snippet: Anti-Human C3a (20C-CR6032RP) was purchased from Fitzgerald and anti-human C5a (AF2037) was purchased from R&D Systems.

Techniques: Incubation, Expressing, Flow Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay, Labeling, Membrane, Isolation, SDS Page, Electrophoresis

Journal: Nature immunology

Article Title: Absent C3a and C5a receptor signaling into CD4 + T cells enables auto-inductive TGF-β1 signaling and induction of Foxp3 + T regulatory cells

doi: 10.1038/ni.2499

Figure Lengend Snippet: Experiments were repeated two times. (a) Flow sorted human CD45RA + CD25 − CD4 + T cells (1×10 6 ) were incubated for 3 days with soluble anti-CD3 mAb (3 μg/ml), rhIL-2 (5 ng/ml), and 2.5×10 5 autologous CD11c + DCs in the absence or presence of 1) TGF-β1 (5 ng/ml), 2) each of C3aR-A/C5aR-A (10 nM), or 3) each of anti-C3a/C5a mAbs (10 μg/ml). Percent Foxp3 + CD25 + CD4 + T cells then were determined by flow cytometry. (b) Flow sorted CD25 + cells from (a) were incubated for 3 days with in differing T eff /iT reg ratios with 1×10 6 CFSE labeled autologous naive CD25 − cells, anti-CD3 mAb (3 μg/ml), and 1×10 4 autologous CD11c + DCs. Percent dividers was determined by CFSE dilution. (c) CD45RA + CD25 − CD4 + T cells (1×10 6 ) were isolated from 5 healthy controls (NC) and 3 MS patients by flow sorting. The cells were incubated for 3 days with soluble anti-CD3 mAb (3 μg/ml), rhIL-2 (5 ng/ml), and 2.5×10 5 autologous DCs in the absence or presence of 1) rhTGF-β1 (5 ng/ml), 2) C3aR-A/C5aR-A (10 nM), or 3) anti-C3a/C5a mAbs (5 μg/ml). Cells were washed and sorted on CD25. After sorting, CD25 + (T reg ) were incubated for 3 days with anti-CD3 mAb, 2.5×10 5 autologous DCs, and 10 6 CD25 − (Effector) cells prelabeled with CFSE. Percent dividers was determined by CFSE dilution.

Article Snippet: Anti-Human C3a (20C-CR6032RP) was purchased from Fitzgerald and anti-human C5a (AF2037) was purchased from R&D Systems.

Techniques: Incubation, Flow Cytometry, Labeling, Isolation

Journal: Immunology

Article Title: Development and characterization of novel anti‐C5 monoclonal antibodies capable of inhibiting complement in multiple species

doi: 10.1111/imm.13083

Figure Lengend Snippet: Functional assays to determine whether monoclonal antibodies (mAb) 4G2, 7D4 and 10B6 inhibit complement in different species. (a–e) Classical pathway haemolysis (CH50). Sera tested were human (a), rat (b), rabbit (c), guinea pig (d) and mouse (e). Commercial mAb RO7112689 and Eculizumab were used as comparators. (f) Calculation of 50% inhibitory dose showed that human C5 inhibition by mAb 10B6 was equivalent to the two comparator mAb, RO7112689 and Eculizumab, and that 4G2 and 7D4 strongly inhibited rat C5. (g, h) Alternative pathway (AP) haemolysis (AH 50 ) assay using human (g) and rat (h) serum; all tested mAb inhibited AP in human serum, while 4G2 and 7D4 inhibited rat AP. (i) Inhibition of C5a generation by the novel mAb in a classical pathway assay with human serum; all three mAb efficiently inhibited C5a generation in a dose‐dependent manner. All experiments were repeated three times with the same results. The error bars are standard errors of triplicates. The dashed lines correspond to the comparator mAb.

Article Snippet: Blots were probed with anti‐C5a mAb (#HM2079; Hycult Biotech), detected using donkey anti‐mouse IgG‐HRP (Jackson ImmunoResearch, 715‐035‐150).

Techniques: Functional Assay, Inhibition

Journal: Immunology

Article Title: Development and characterization of novel anti‐C5 monoclonal antibodies capable of inhibiting complement in multiple species

doi: 10.1111/imm.13083

Figure Lengend Snippet: Impact of anti‐C5 monoclonal antibodies (mAb) on cleavage of C5 by neutrophil elastase. (a) Western blot of atypical cleavage of C5 by neutrophil elastase (NE). C5 was mixed with each mAb at 5× molar excess in HEPES‐buffered saline (HBS), NE was added at 420 n m , incubated and the reaction was stopped by addition of protease inhibitors. Samples (1 μg) were resolved on 4–20% SDS–PAGE gels under reducing (R) conditions and processed for WB to detect intact or cleaved C5 and C5a using an anti‐C5a mAb that also detects native C5 α chain (Hycult; mAb 2942). C5 α (115 000 MW), C5a (10·4 000 MW), CI; proteases cocktail inhibitors, Ecul.; Eculizumab, RO; RO7112689, 3D3; irrelevant non‐blocking anti‐C5 mAb. Note that numerous unidentified C5 cleavage products are present in all NE‐treated samples. (b) Densitometry analysis of the C5a band using imagej (expressed as % relative to the C5a generation in the absence of antibody; 100%) confirmed that mAb 4G2 and commercial mAb RO7112689 efficiently inhibited generation of C5a. (c) measurement of C5a generation by ELISA confirmed that mAb 4G2 and RO7112689 inhibited C5a generation while other mAb inhibited weakly. ELISA results are presented as percentage relative to C5a generation in the absence of any mAb (set as 100%; measured as 670 ng/ml in the ELISA). Results are representative of three independent experiments. The error bars are standard errors of triplicates.

Article Snippet: Blots were probed with anti‐C5a mAb (#HM2079; Hycult Biotech), detected using donkey anti‐mouse IgG‐HRP (Jackson ImmunoResearch, 715‐035‐150).

Techniques: Western Blot, Incubation, SDS Page, Blocking Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Intrinsic bias at non-canonical, β-arrestin-coupled seven transmembrane receptors

doi: 10.1101/2021.02.02.429298

Figure Lengend Snippet: A. Schematic representation of canonical GPCR and non-canonical 7TMR pairs activated by a common agonist. C5a, complement C5a; C5aR1, C5a receptor subtype 1; C5aR2, C5a receptor subtype 2; CCL7, chemokine CCL7; CCR2, C-C chemokine receptor subtype 2; D6R, decoy D6 receptor. B-C. Agonist-induced dissociation of heterotrimeric G-proteins for C5aR1-C5aR2, and CCR2-D6R pairs measured using NanoBiT complementation assay. HEK-293 cells expressing the indicated receptor and Sm/Lg-BiT constructs of G-protein α,β,γ sub-units were stimulated with corresponding ligands, and the change in luminescence signal upon NanoBiT dissociation was measured as a readout of G-protein coupling. Data represent three independent experiments, normalized with respect to baseline signal (i.e. vehicle treatment). D. Agonist-induced second messenger response measured using GloSensor assay (cAMP stimulation, and inhibition of forskolin-induced cAMP level), and Fluo-4 NW calcium mobilization assay. HEK-293 cells expressing the indicated receptor were used for these assays using standard protocols as described in the method section. For each of the second messenger assay, a well-established prototypical GPCR was included as a reference (V 2 R, vasopressin receptor subtype 2; B 2 R, bradykinin receptor subtype 2). Data are normalized with respect to maximum signal (treated as 100%) and represent mean±sem of four independent experiments.

Article Snippet: Wild-type (WT), C5aR1−/− and C5aR2−/− mice on a C57BL/6J genetic background (n=5-15) were administered with recombinant mouse C5a (Sino Biological, China) at a dose of 50 μg kg-1 via intravenous injection (tail vein).

Techniques: Expressing, Construct, Inhibition, Calcium Mobilization Assay

Journal: bioRxiv

Article Title: Intrinsic bias at non-canonical, β-arrestin-coupled seven transmembrane receptors

doi: 10.1101/2021.02.02.429298

Figure Lengend Snippet: A. HEK-293 cells expressing the indicated receptor constructs were stimulated with agonist, lysed and incubated with purified βarr1/2. Subsequently, the receptor was immunoprecipitated using anti-FLAG M1 antibody agarose and co-purified βarrs were detected using Western blotting. A representative blot from three independent experiments is shown here. B-C. Agonist-induced trafficking of βarrs was measured in HEK-293 cells expressing the indicated receptor constructs and mYFP-tagged βarrs using live cell confocal microscopy. Cells were stimulated with saturating concentration of agonists (CCL7, 100 nM; C5a,100 nM) and trafficking of βarrs was monitored at indicated time-points. Representative images from three independent experiments are shown (scale bar is 10μm). D. Internalized D6R and C5aR2 co-localize with βarr2 as monitored by confocal microscopy. HEK-293 cells expressing the indicated receptor and βarr2-mYFP were stimulated with agonist, followed by fixation, permeabilization and staining of the receptor using DyLight-688 conjugated anti-FLAG M1 antibody. Localization of the receptor and βarr2 was visualized using confocal microscopy, and representative images from three independent experiments are shown (scale bar is 10μm). The Pearson’s Correlation Coefficient (PCC) were 0.68±0.05, 0.72±0.06, 0.94±0.01 and 0.96±0.01 for C5aR2, 0min (12 cells), C5aR2, 10min (13 cells), D6R, 0min (14 cells) and D6R, 10min (15 cells), respectively. E. Single particle analysis of C5aR2-V2-βarr1-Fab30 complex further corroborates the interaction of βarr1 with C5aR2. Sf 9 cells expressing a chimeric C5aR2 construct (C5aR2-V2), GRK2 CAAX and βarr1 were stimulated with C5a (100 nM), stabilized using Fab30, followed by affinity purification of the complex on anti-Flag M1 column. Subsequently, the fractions containing the complex were further isolated by size-exclusion chromatography and subjected to negative staining based single particle analysis. 2D-class averages based on approximately ten thousand particles are shown here, and a typical 2D class average is indicated together with a schematic representation of the complex.

Article Snippet: Wild-type (WT), C5aR1−/− and C5aR2−/− mice on a C57BL/6J genetic background (n=5-15) were administered with recombinant mouse C5a (Sino Biological, China) at a dose of 50 μg kg-1 via intravenous injection (tail vein).

Techniques: Expressing, Construct, Incubation, Purification, Immunoprecipitation, Western Blot, Confocal Microscopy, Concentration Assay, Staining, Single Particle, Affinity Purification, Isolation, Size-exclusion Chromatography, Negative Staining

Journal: bioRxiv

Article Title: Intrinsic bias at non-canonical, β-arrestin-coupled seven transmembrane receptors

doi: 10.1101/2021.02.02.429298

Figure Lengend Snippet: A. CCL7 stimulation leads to robust ERK1/2 phosphorylation in HEK-293 cells expressing CCR2, however, it fails to elicit any detectable ERK1/2 phosphorylation for D6R as measured by Western blotting. B. C5aR1 stimulation exhibits a typical ERK1/2 phosphorylation pattern upon agonist-stimulation while C5aR2 cells display an elevated level of basal ERK1/2 phosphorylation, which decreases upon C5a-stimulation. C. PTX-treatment inhibits C5a-induced ERK1/2 phosphorylation downstream of C5aR1 but it fails to inhibit the elevated level of basal ERK1/2 phosphorylation for C5aR2. D. Treatment of cells with U0126, a MEK-inhibitor completely abolishes ERK1/2 phosphorylation for both, C5aR1 and C5aR2 suggesting the involvement of a canonical mechanism of ERK1/2 phosphorylation. The right panels show quantification based on densitometry data from 4-6 experiments analyzed using One- or Two-Way ANOVA (*p<0.05, **p<0.01, ***p<0.001, ***p<0.0001).

Article Snippet: Wild-type (WT), C5aR1−/− and C5aR2−/− mice on a C57BL/6J genetic background (n=5-15) were administered with recombinant mouse C5a (Sino Biological, China) at a dose of 50 μg kg-1 via intravenous injection (tail vein).

Techniques: Expressing, Western Blot

Journal: bioRxiv

Article Title: Intrinsic bias at non-canonical, β-arrestin-coupled seven transmembrane receptors

doi: 10.1101/2021.02.02.429298

Figure Lengend Snippet: A. Phospho-antibody array on HEK-293 cells expressing C5aR1 or C5aR2 reveals phosphorylation/dephosphorylation of a few common and several distinct cellular proteins. Of these, C5a-stiulation of C5aR2-expressing HEK-293 cells exhibits robust enhancement of p90RSK phosphorylation, which is also common to C5aR1. B. C5a-stimulation of C5aR2 expressing HEK-293 cells is validated using Western blotting, which validates the phospho-antibody array data. A representative blot from six experiments and densitometry-based quantification (average±sem) is presented (*p<0.05, ***p<0.001; One-Way ANOVA). C. C5a-stiulated p90RSK phosphorylation is βarr1 dependent as knock-down of βarr1 in HEK-293 cells expressing C5aR2 reduces p90RSK phosphorylation. A representative blot from five experiments and densitometry-based quantification (average±sem) is presented (**p<0.01; One-Way ANOVA). D. Stimulation of human macrophage derived monocytes (hMDMs) with either C5a or P32 (a C5aR2-selective agonist) results in significant p90RSK phosphorylation. Importantly, PMX53, a C5aR1-selective antagonist, does not block p90RSK phosphorylation suggesting that it is mediated by C5aR2. E. A significant component of C5a-induced polymorphonuclear leukocyte (PMN) mobilization depends on C5aR2. WT, C5aR1 −/− and C5aR2 −/− mice on a C57BL/6J genetic background (n = 5-15) were intravenously administered with recombinant mouse C5a (50 μg kg −1 ). Tail tip collected blood was smeared onto a slide followed by staining and counting of white blood cells, with the proportion of PMNs calculated. Data is presented as average±sem (*p<0.05, ***p<0.001; One- and Two-Way ANOVA).

Article Snippet: Wild-type (WT), C5aR1−/− and C5aR2−/− mice on a C57BL/6J genetic background (n=5-15) were administered with recombinant mouse C5a (Sino Biological, China) at a dose of 50 μg kg-1 via intravenous injection (tail vein).

Techniques: Ab Array, Expressing, De-Phosphorylation Assay, Western Blot, Derivative Assay, Blocking Assay, Recombinant, Staining