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: The Journal of biological chemistry

Article Title: Inhibition of cleavage of human complement component C5 and the R885H C5 variant by two distinct high affinity anti-C5 nanobodies.

doi: 10.1016/j.jbc.2023.104956

Figure Lengend Snippet: Figure 3. Cryo-EM structures of UNbC5-1 and UNbC5-2 in complex with C5. A, cryo-EM density map of the C5:UNbC5-1:UNbC5-2 complex at 3.6 Å resolution is depicted with C5 colored in gray, UNbC5-1 in green, and UNbC5-2 in orange. B, comparison of the C5 structure excluding the C345c domain (3CU7, blue) to the C5 of the C5:UNbC5-1:UNbC5-2 structure (gray). Left panel shows both structures in cartoon representation, superimposed and aligned by the C5 MG-ring. Domains C5a, C5d, and CUB of the C5:UNbC5-1:UNbC5-2 structure shows a displacement when compared to the C5 structure. Right panel shows a zoom in of the domain C5a reoriented and aligned, both structures with residue R751 showed as sticks in orange for the C5:UNbC5-1:UNbC5- 2 structure and in cyan for C5 (3CU7). C–E, zoom in on the C5 interface with nanobodies UNbC5-1 (C and D), and UNbC5-2 (E), in the same colors introduced in panel A. Amino acids that likely contribute to the interface are shown as sticks representation and annotated. Data information: (A), cryo-EM density map visualized in ChimeraX and (B–E) figures produced in PyMOL from refined structure. CUB, complement C1r/C1s, Uegf, Bmp1; MG, macroglobulin.

Article Snippet: To measure C5a formation, 25 μl of the supernatant was diluted with 25 μl 1% BSA PBS-T and added to a Nunc Maxisorp plate coated with 1 μg/ml anti-C5a capture antibody (C5a DuoSet ELISA kit, R&D systems).

Techniques: Cryo-EM Sample Prep, Comparison, Residue, Produced

Journal: The Journal of biological chemistry

Article Title: Inhibition of cleavage of human complement component C5 and the R885H C5 variant by two distinct high affinity anti-C5 nanobodies.

doi: 10.1016/j.jbc.2023.104956

Figure Lengend Snippet: Figure 5. UNbC5-1 competes with eculizumab in binding to the MG7 domain of C5. A, superposition of the C5:UNbC5-1:UNbC5-2 and the C5- eculizumab-Fab complex structure. Surface representation shows epitope overlap between UNbC5-1 and eculizumab-Fab. UNbC5-1 is shown in green, UNbC5-2 in orange, eculizumab-Fab in blue, most domains of C5 in light gray, and domain MG7 in dark gray. B, left panel shows a 45 rotation of panel A. Right panel shows a zoom in of the epitope of UNbC5-1 (green) and eculizumab-Fab (blue) on the MG7 domain (dark gray) with an extensive overlap (pink) of the epitope between the interface of both molecules in C5. The overlapping amino acids for both interfaces are indicated, with residue R885 in bold. C, competition ELISA with C5-coated microtiter plates incubated with 300 nM UNbC5-1 and a titration of Ecu-mab or RaCI3 (5.49–4000 nM, 3-fold). Binding of UNbC5-1 to C5 was measured using polyclonal rabbit-anti-VHH QE19 antibodies and donkey-anti-rabbit-HRP antibodies at A450 nm. Data information: (A and B) figures produced in PyMOL, (C), data represent mean ± SD of four individual experiments. MG, macroglobulin.

Article Snippet: To measure C5a formation, 25 μl of the supernatant was diluted with 25 μl 1% BSA PBS-T and added to a Nunc Maxisorp plate coated with 1 μg/ml anti-C5a capture antibody (C5a DuoSet ELISA kit, R&D systems).

Techniques: Binding Assay, Residue, Enzyme-linked Immunosorbent Assay, Incubation, Titration, Produced

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: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Generation of Multiple Fluid-Phase C3b:Plasma Protein Complexes During Complement Activation. Possible Implications in C3 Glomerulopathies

doi: 10.4049/jimmunol.1302288

Figure Lengend Snippet: Pooled normal human serum or citrated plasma were activated at 37°C with 416 U/ml CVF for the indicated times. In addition to CVF, 2 mM Mg2+ was added to plasma to overcome the chelation effects of sodium citrate. At each time-point, activation was stopped by placing the sample on ice. Panel A: Aliquots were separated on an 8% SDS-PAGE and then immunoblotted for the indicated proteins. Gels were cropped to focus only on the high molecular weight SDS-resistant bands. Samples also were blotted for factor B to verify cleavage as an indicator of complement activation. The 75 kDa β-chain of C3 was utilized as a loading control. Panel B: C5a ELISA of complement activation in serum. Panel C: C5a ELISA of complement activation in citrated plasma.

Article Snippet: Briefly, Maxi-sorb 96 well plates were coated with 500 ng of mouse monoclonal anti-human C5a (clone 295003; R&D Systems, Minneapolis, MN) capture antibody at 4°C overnight.

Techniques: Clinical Proteomics, Activation Assay, SDS Page, High Molecular Weight, Control, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Generation of Multiple Fluid-Phase C3b:Plasma Protein Complexes During Complement Activation. Possible Implications in C3 Glomerulopathies

doi: 10.4049/jimmunol.1302288

Figure Lengend Snippet: Panel A: The amount of C5a generated in reconstituted C3-depleted serum samples measured by ELISA. Antibody coated sheep erythrocytes were treated with either C3-depleted serum alone or C3-depleted serum reconstituted with 1.3 mg/ml native C3, hydroxylamine treated C3 (C3-NH2OH) or C3b and incubated at 37°C for 1 hour. The supernatant was removed and C5a levels quantified by C5a ELISA. Panel B: C3-depleted serum alone or reconstituted with native C3 or hydroxylamine treated C3 (C3-NH2OH), both at 1.3 mg/ml, were activated with CVF (416 U/ml) plus 0.4 mM Mg2+ for 15 minutes at 37°C. CVF activated normal human serum was included as a positive control. Aliquots were separated using 8% SDS-PAGE and immunoblotted for DBP, α1PI, α1AG, factor B and C3. Panel C: Pooled normal human serum was sham-treated with PBS (lane 1) or complement was activated by incubating serum at 37°C using either 416 U/ml CVF (lane 2), 10 mg/ml zymosan A (lane 3), 0.5 mg/300 μl heat-aggregated human IgG (lane 4). As a control to inhibit complement activation, 10 mM EDTA was added to serum prior to addition of CVF (lane 5). Serum aliquots were separated on an 8% SDS-PAGE and then immunoblotted for C3. Panel D: Pooled normal human serum or citrated plasma were activated at 37°C with 416 U/ml CVF for the indicated times. In addition to CVF, 2 mM Mg2+ was added to plasma to overcome the chelation effects of sodium citrate. At each time-point, activation was stopped by placing the sample on ice. Aliquots were separated on an 8% SDS-PAGE and then immunoblotted C3.

Article Snippet: Briefly, Maxi-sorb 96 well plates were coated with 500 ng of mouse monoclonal anti-human C5a (clone 295003; R&D Systems, Minneapolis, MN) capture antibody at 4°C overnight.

Techniques: Generated, Enzyme-linked Immunosorbent Assay, Incubation, Positive Control, SDS Page, Control, Activation Assay, Clinical Proteomics

Journal: The FASEB Journal

Article Title: IL-17A deficiency mitigates bleomycin-induced complement activation during lung fibrosis

doi: 10.1096/fj.201700289R

Figure Lengend Snippet: IL-17A activates C′ by modulating the mRNA and protein expression of the C′ components in vitro. A) Normal human primary AECs were exposed to recombinant human IL-17A (100 ng/ml; 24 h). Subsequent to RNA isolation, cDNA was generated and subjected to real-time quantitative PCR analyses. Means ± sem; Student’s t test. B) Protein lysates from AECs exposed to recombinant human IL-17A (100 ng/ml; 4 h) were subjected to immunoblot analysis against C3aR, C5aR, DAF, and vinculin. C) Conditioned medium collected from AECs exposed to the indicated doses of IL-17A for 4 h was analyzed for active C3a by ELISA. Means ± sem. D) AECs exposed to IL-17A (100 ng/ml; 4 h) were methanol fixed, immunostained for DAF, and counterstained with DAPI. Results represent 3 independent experiments. Scale bars, 100 µm.

Article Snippet: The antibodies used for immunoblot analysis or immunofluorescent labeling are as follows: decay-accelerating factor (DAF)-clone H-319 (sc-9156); and vinculin-clone7F9 (sc-73614) (Santa Cruz Biotechnology), C3a receptor (C3aR; NBP1-61567), and C5a receptor (C5aR; NBP2-15649; Novus Biologicals, Littleton, CO, USA).

Techniques: Expressing, In Vitro, Recombinant, Isolation, Generated, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: The FASEB Journal

Article Title: IL-17A deficiency mitigates bleomycin-induced complement activation during lung fibrosis

doi: 10.1096/fj.201700289R

Figure Lengend Snippet: IL-17A deficiency protects against BLEO-induced local C′ activation. The BALF samples collected for the analysis shown in Fig. 2 were also analyzed for C3a (A), C5a (B), and C5b-9 (C) levels by ELISA (n = 7–8/group). Means ± sem. One-way ANOVA, Newman-Keuls (A, C), and Bonferroni (B).

Article Snippet: The antibodies used for immunoblot analysis or immunofluorescent labeling are as follows: decay-accelerating factor (DAF)-clone H-319 (sc-9156); and vinculin-clone7F9 (sc-73614) (Santa Cruz Biotechnology), C3a receptor (C3aR; NBP1-61567), and C5a receptor (C5aR; NBP2-15649; Novus Biologicals, Littleton, CO, USA).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay