complement c3 protein  (MedChemExpress)

Journal: bioRxiv

Article Title: Loss of Complement Factor D suppresses alternative pathway activation but fails to reduce lipofuscin accumulation in the retinal pigmented epithelium of Abca4 -/- mice

doi: 10.1101/2025.10.25.684461

Figure Lengend Snippet: Immunofluorescence labeling of PFA-fixed wild type and C3 -/- mouse eyecup tissue with A) anti-C3 (MP Biomedicals, 55463) and B) anti-C3d (R&D Systems, AF2655) antibodies, showing genuine antibody labeling in the RPE of wild type samples, localized primarily to the basal side of the RPE. Note suspected non- specific binding of the anti-C3 primary antibody to photoreceptor outer segments (OS). Abca4 -/- samples were included as a no primary control, to show general tissue AF. Labeling using the anti-C3 antibody was performed using frozen sections and labeling using the anti-C3d antibody was performed using paraffin-embedded tissue sections. General tissue AF at 488 nm was included to assist in identifying eyecup structures and for comparison to immunofluorescence signal in the no primary control conditions. Note that each C3 antibody may detect multiple C3 fragments. The scale bar represents 10 µm and is consistent across all images. Choroid, RPE and OS are shown. Arrowheads show red blood cells, which are fluorescent at 488 nm in paraffin sections. Arrows show the location of suspected RPE basal labyrinth.

Article Snippet: C3 labeling was quantified in mouse eyecup structures, such as the RPE and choroid, using antibodies generated against full-length mouse C3 protein (referred to as “anti-C3”, MP Biomedicals) or purified mouse C3d (referred to as “anti-C3d”, R&D Systems) ( ).

Techniques: Immunofluorescence, Labeling, Antibody Labeling, Binding Assay, Control, Comparison

Journal: bioRxiv

Article Title: Loss of Complement Factor D suppresses alternative pathway activation but fails to reduce lipofuscin accumulation in the retinal pigmented epithelium of Abca4 -/- mice

doi: 10.1101/2025.10.25.684461

Figure Lengend Snippet: A) Representative images showing immunofluorescence labelling in PFA-fixed wild type, Abca4 -/- , Cfd -/- and Abca4 -/- ; Cfd -/- mouse eyecup tissue sections, using anti-C3 (MP Biomedicals) and anti-C3d (R&D Systems) antibodies. Anti-C3 immunofluorescence was performed using 6-12-mo frozen sections and anti-C3d immunofluorescence was performed using 12-mo paraffin embedded sections. For each image, 488 nm AF (green) and C3 immunofluorescence (magenta) are shown together, to aid in localizing the immunofluorescence signal. C3 immunofluorescence is also shown on its own in greyscale. Choroid, RPE and photoreceptor outer segments (OS) are labelled, with dotted lines indicating the basal (top) and apical (bottom) boundaries of the RPE. Arrowheads show the location of Bruch’s membrane, between the RPE basal labyrinth and the choroid region. Scale bar represents 10 µm. B-C) Quantification of total RPE immunofluorescence signal in each genotype using the anti-C3 and anti-C3d antibodies, respectively. Total immunofluorescence was quantified by measuring the mean immunofluorescence signal in the RPE of 3-4 images minus the fluorescent signal in 3-4 no primary antibody control images, per mouse. Each data point represents one mouse. Mean +/- 95% C.I. are shown. Data were analyzed by 1-way ANOVA with Tukey’s multiple comparisons test. **** indicates p<=0.0001

Article Snippet: C3 labeling was quantified in mouse eyecup structures, such as the RPE and choroid, using antibodies generated against full-length mouse C3 protein (referred to as “anti-C3”, MP Biomedicals) or purified mouse C3d (referred to as “anti-C3d”, R&D Systems) ( ).

Techniques: Immunofluorescence, Membrane, Control

Journal: bioRxiv

Article Title: Loss of Complement Factor D suppresses alternative pathway activation but fails to reduce lipofuscin accumulation in the retinal pigmented epithelium of Abca4 -/- mice

doi: 10.1101/2025.10.25.684461

Figure Lengend Snippet: A) Schematic representation of production and breakdown of the C3 protein. Estimated fragment molecular weights under reducing conditions are shown. Western blot was performed using eyecup lysate (RPE, choroid, sclera) detected using an antibody made against the C3d region (anti-C3d). Yellow shows the C3d region of the protein, which is part of the C3 α-chain (C3α). Note that the C3d region of the C3 protein contains the thioester site, which participates in covalent binding to target molecules (yellow arrowheads), potentially affecting fragment molecular weight of the C3b, iC3b, C3dg and C3d α-chain. B) Qualitative western blot images showing labelling of wild type and C3 -/- eyecup tissue using the anti-C3d antibody. Marked regions i-vi indicate C3 fragments of interest and are shown zoomed-in in greyscale in panel (C) with predicted C3 fragment identities indicated.

Article Snippet: C3 labeling was quantified in mouse eyecup structures, such as the RPE and choroid, using antibodies generated against full-length mouse C3 protein (referred to as “anti-C3”, MP Biomedicals) or purified mouse C3d (referred to as “anti-C3d”, R&D Systems) ( ).

Techniques: Western Blot, Binding Assay, Molecular Weight

Journal: bioRxiv

Article Title: Loss of Complement Factor D suppresses alternative pathway activation but fails to reduce lipofuscin accumulation in the retinal pigmented epithelium of Abca4 -/- mice

doi: 10.1101/2025.10.25.684461

Figure Lengend Snippet: A) Representative blots labelled with the anti-C3d antibody, showing all genotypes. Note the faint C3 α-chain fragment detected with the anti-C3d antibody, which may be the opsonized iC3b α62 fragment (ops iC3b) and was frequently detected in Cfd +/+ but not Cfd -/- conditions. Arrowhead (*) indicates the suspected opsonized C3b α-chain fragment selected for quantification. Higher molecular weight opsonized C3bα fragments were not quantified due to transfer inconsistencies and blot damage, which frequently affected the high molecular weight region of the blots. Rpe65 and β-actin were included as loading controls. B) Quantification of various C3 fragments under reducing conditions from mouse eyecup lysate, detected by the anti-C3d antibody. Animals used in this experiment range from 6-12 months of age. Relative quantities of C3 preprotein, C3α, iC3(H2O) α72, opsonized C3bα (*), iC3b α62 and C3dg were normalized to Rpe65 and β-actin, which were shown to have stable signal across the experimental conditions of interest (Supplementary Figure 6). Given the large number of experimental conditions, samples were run across multiple blots with a mix of genotypes per blot. To control for variability between blots, normalized C3 fragment quantities were expressed relative to the C3α fragment from WT samples. Note that quantities of C3α and iC3(H2O) α72 (filled circles) are represented on the left y-axis, and quantities of the preprotein, opsonized C3bα, iC3b α62 and C3dg fragments (empty circles) are represented on the right y-axis. Data were analyzed using a two-way ANOVA, revealing an effect of C3 fragment (p<0.0001), genotype (p<0.0001) and C3 fragment X genotype (p<0.0001). Multiple comparisons were performed using Tukey’s test, and p-values <0.05 are shown on the graph. C-D) Analysis of data from D, organized by independent variable (Abca4 genotype and Cfd genotype, respectively). Within each C3 fragment, data are represented relative to the respective control conditions ( Abca4 +/+ or Cfd +/+ ). Data were analyzed using a two-way ANOVA, revealing an effect of C3 fragment X genotype for the Cfd genotype (p<0.0001) but not Abca4 genotype (p=0.6). Multiple comparisons were performed using Šídák’s test, and p-values <0.05 are shown on the graph. Each data point is a measurement from a single animal, and data are represented as mean +/- 95% C.I. for all graphs.

Article Snippet: C3 labeling was quantified in mouse eyecup structures, such as the RPE and choroid, using antibodies generated against full-length mouse C3 protein (referred to as “anti-C3”, MP Biomedicals) or purified mouse C3d (referred to as “anti-C3d”, R&D Systems) ( ).

Techniques: Molecular Weight, High Molecular Weight, Control

Journal: Cells

Article Title: House Dust Mite Nebulization Drives Alarmin and Complement Activation in a Murine Tracheal Air–Liquid Interface Culture System

doi: 10.3390/cells14201598

Figure Lengend Snippet: Time-dependent complement activation and anaphylatoxin receptor expression in ALI cultures exposed to HDM. ( a ) C3 and C3a concentrations in cell lysates and the SN of unstimulated (steady-state) ALI cultures and 24, 48 and 72 h after HDM exposure (steady-state, 24, 48 and 72 h: n = 6). * p < 0.05; ** p < 0.01; *** p < 0.001. ( b ) HDM and HI-HDM mediated cleavage of hC3 into hC3a ( n = 3 independent experiments). Data are shown as mean ± SEM. Data were analyzed using an unpaired T -test. *** p < 0.001. ( c ) C5 and C5a concentrations in cell lysates and the SN of steady-state ALI cultures and 24, 48 and 72 h after HDM exposure. Data are shown as mean ± SEM (steady-state, 24, 48 and 72 h: n = 6). ( d ) Comparison of C3 and C5 ( left panel ), C3a and C5a ( right panel ) concentrations in cell lysates from steady-state ALI cultures and 24, 48 and 72 h after HDM exposure. Data are shown as mean ± SEM (steady-state, 24, 48 and 72 h: n = 6). Data were analyzed using two-way ANOVA with Šidák’s multiple comparisons test. **** p < 0.0001. ( e ) Immunofluorescence analysis of C3aR and C5aR1 expression in unstimulated ALI cultures and 24, 48 and 72 h after HDM exposure. Data are shown as mean ± SEM ( n = 3). Data were analyzed using one-way ANOVA with Holm–Šidák’s multiple comparisons test. * p < 0.05; ** p < 0.01. The number of experiments shown in ( a , c – e ) refers to biological replicates derived from 6 independent preparations of trachea, each representing a separate cell isolation and culture.

Article Snippet: Plates were washed four times with 0.05% Tween 20 in PBS; serial dilutions of recombinant mouse C3a (R&D Systems #8085-C3-025—Minneapolis, MN, USA) were performed; and samples were added (25 μL/well) and incubated at RT for 90 min. After washing four times with 0.05% Tween 20 in PBS, 25 μL/well of 1 μg/mL in 1% BSA/PBS biotinylated anti-mouse C3a detection antibody (clone I87-419, BD Biosciences #558251—San Jose, CA, USA) was added and incubated for 1 h at RT.

Techniques: Activation Assay, Expressing, Comparison, Immunofluorescence, Derivative Assay, Cell Isolation

Journal: The Journal of Clinical Investigation

Article Title: Urine proteins reveal distinct coagulation and complement cascades underlying acute versus chronic lupus nephritis

doi: 10.1172/JCI186143

Figure Lengend Snippet: ( A ) The blood coagulation cascade, highlighting molecules whose levels in urine are significantly correlated with renal pathology AI (blue font), CI (CI) (red font), or both (purple font) in LN. Other proteins are listed in black font (interrogated but not significantly changed) or grey font (not interrogated by the proteomic screen). Uninterrupted arrows indicate activation, and interrupted arrows signify inhibition or cleavage of downstream protein or substrate. The yellow bubbles highlight only proteins significantly elevated in LN with high-CI versus low-CI (FC ≥ 2; P < 0.05) or higher in CI than in AI by at least 10%. The pink bubbles highlight proteins whose levels were significantly elevated only in patients with LN with high-AI versus low-AI (FC ≥ 2; P < 0.05) or higher in AI than in CI by at least 10% (in terms of correlation coefficient or FC). Also shown is a Spearman correlation heatmap displaying correlations among the 27 coagulation-related proteins and renal pathology metrics. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. ( B ) The complement activation pathway highlighting molecules whose levels in urine are significantly elevated with AI, CI, or both. See A for other annotation details. Also shown is a Spearman correlation heatmap displaying correlations among the 32 complement related proteins and their paired renal pathology metrics, as detailed in A . α2-AP, MG, α2-antiplasmin, α2-macroglobulin; APC, activated protein C; AT, antithrombin; B, factor B; BK, bradykinin; C1 INH, C1 esterase inhibitor; C4BP, C4 binding protein; CL-K1, collectin kidney 1; D, factor D; DAF, decay-accelerating factor; FDP, fibrin degradation products; H, factor H; I, factor I; MAP-1, MBL/ficolin-associated protein 1; MASP, mannan-binding lectin-associated serine protease; MBL, mannose-binding lectin; MCP, membrane cofactor protein; P, properdin; PK, prekallikrein; sMAP, small MBL-associated protein; TM, thrombomodulin; tPA, tissue plasminogen activator; uPA, urokinase.

Article Snippet: After 3 days of differentiation, the medium was replaced with serum-free medium for 24 hours, after which the cells were treated for 72 hours with either vehicle or 10 ng/mL C3a (R&D Systems 3677-C3-025) or 10 ng/mL of C5a (R&D Systems 2037-C5-025/CF).

Techniques: Coagulation, Activation Assay, Inhibition, Binding Assay, Membrane

Journal: The Journal of Clinical Investigation

Article Title: Urine proteins reveal distinct coagulation and complement cascades underlying acute versus chronic lupus nephritis

doi: 10.1172/JCI186143

Figure Lengend Snippet: ( A ) Shown are representative fields from 3 independent experiments. Complement proteins C3a and C5a increased the expression of ECM proteins in THP1 macrophages, BMDMs, and HK2 proximal tubule cells after 72 hours of treatment in serum-free medium. Scale bars: 50 μm. ( B – D ) The scatter plots show the mean staining intensity per THP-1 macrophage ( B ), BMDM ( C ), and HK2 proximal tubule cell ( D ), normalized to expression levels in their respective vehicle-treated groups. Each data point corresponds to quantified fluorescence intensity in a single field of view (FOV) from the microscope, and the larger dots represent the average of FOVs in biological replicates, each of which is color coded. RT-qPCR analysis of ECM protein coding genes were measured in BMDMs ( E ) and in HK2 proximal tubule cells ( F ). Gene expression was normalized to the expression of 18S ribosomal RNA in the same sample and then normalized to the expression level of vehicle-treated group ( n = 4). * P < 0.05, ** P < 0.01, and *** P < 0.001 by 2-tailed Student’s t test.

Article Snippet: After 3 days of differentiation, the medium was replaced with serum-free medium for 24 hours, after which the cells were treated for 72 hours with either vehicle or 10 ng/mL C3a (R&D Systems 3677-C3-025) or 10 ng/mL of C5a (R&D Systems 2037-C5-025/CF).

Techniques: Expressing, Staining, Fluorescence, Microscopy, Quantitative RT-PCR, Gene Expression

Journal: The Journal of Clinical Investigation

Article Title: Urine proteins reveal distinct coagulation and complement cascades underlying acute versus chronic lupus nephritis

doi: 10.1172/JCI186143

Figure Lengend Snippet: Circulating immune complexes and Abs planted directly within glomerular and tubulo-interstitial regions of the kidneys may fix complement, resulting in complement activation. The alternative pathway may further amplify complement activation within the kidneys. The products of C3 and C5 convertases, including the anaphylatoxins C3a and C5a, engage cognate receptors on a wide spectrum of immune cells, leading to immune cell activation, release of cytokines and chemokines, and acute inflammation, leading to high AI, as depicted on the left. Long-standing, unresolved complement activation and eventual formation of MAC may additionally engage and activate more immune and renal-resident cells, leading to tissue damage and repair, ECM deposition, and renal fibrosis, leading to high CI, as depicted on the right.

Article Snippet: After 3 days of differentiation, the medium was replaced with serum-free medium for 24 hours, after which the cells were treated for 72 hours with either vehicle or 10 ng/mL C3a (R&D Systems 3677-C3-025) or 10 ng/mL of C5a (R&D Systems 2037-C5-025/CF).

Techniques: Activation Assay