apolipoprotein ciii  (Elabscience Biotechnology)

Journal: Blood Vessels, Thrombosis & Hemostasis

Article Title: A second generation of C3 humanized rats for preclinical evaluation of human C3 inhibitors

doi: 10.1016/j.bvth.2026.100138

Figure Lengend Snippet: The second-generation hC3 rats express human, but not rat C3 in their plasma and tissues, at levels higher than those in the first-generation hC3 rats. (A) Plasma from both human and rat samples were collected, and the presence of human (hC3) and rat C3 (rC3) were evaluated by western blots. Purified human C3 (hC3), rat C3 (rC3), human C3 depleted serum (hC3-dpl), and C3 knockout rat serum rat plasma (rC3-KO) were used as controls. Arrows pointed to the human or rat C3 band. (B) Plasma hC3 levels in the second-generation hC3 rats (both male and female) were evaluated using a human C3 ELISA kit (Hycult) and compared to the levels in the first-generation hC3 rat plasma. (C) Total RNA was extracted from the liver, retina, spleen, kidney, and brain of WT, second-generation, and first-generation hC3 rats, and then semiquantitative reverse transcription PCR was used to detect and quantitate levels of human C3 (hC3) transcripts using the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal controls. 1st gen, first generation; 2nd gen, second generation.

Article Snippet: Blood levels of human C3 protein were measured in the identified human C3 Tg rats using an enzyme-linked immunosorbent assay (ELISA) kit (Hycult, catalog no. HK366) to identify the founder rats with close-to-physiological expression levels of human C3.

Techniques: Clinical Proteomics, Western Blot, Purification, Knock-Out, Enzyme-linked Immunosorbent Assay, Reverse Transcription

Journal: Blood Vessels, Thrombosis & Hemostasis

Article Title: A second generation of C3 humanized rats for preclinical evaluation of human C3 inhibitors

doi: 10.1016/j.bvth.2026.100138

Figure Lengend Snippet: The second-generation hC3 rats express human, but not rat C3 in their plasma and tissues, at levels higher than those in the first-generation hC3 rats. (A) Plasma from both human and rat samples were collected, and the presence of human (hC3) and rat C3 (rC3) were evaluated by western blots. Purified human C3 (hC3), rat C3 (rC3), human C3 depleted serum (hC3-dpl), and C3 knockout rat serum rat plasma (rC3-KO) were used as controls. Arrows pointed to the human or rat C3 band. (B) Plasma hC3 levels in the second-generation hC3 rats (both male and female) were evaluated using a human C3 ELISA kit (Hycult) and compared to the levels in the first-generation hC3 rat plasma. (C) Total RNA was extracted from the liver, retina, spleen, kidney, and brain of WT, second-generation, and first-generation hC3 rats, and then semiquantitative reverse transcription PCR was used to detect and quantitate levels of human C3 (hC3) transcripts using the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal controls. 1st gen, first generation; 2nd gen, second generation.

Article Snippet: Arrows pointed to the human or rat C3 band. (B) Plasma hC3 levels in the second-generation hC3 rats (both male and female) were evaluated using a human C3 ELISA kit (Hycult) and compared to the levels in the first-generation hC3 rat plasma. (C) Total RNA was extracted from the liver, retina, spleen, kidney, and brain of WT, second-generation, and first-generation hC3 rats, and then semiquantitative reverse transcription PCR was used to detect and quantitate levels of human C3 (hC3) transcripts using the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal controls.

Techniques: Clinical Proteomics, Western Blot, Purification, Knock-Out, Enzyme-linked Immunosorbent Assay, Reverse Transcription

Journal: bioRxiv

Article Title: Complement Dysregulation During the Early Phases of Synucleinopathy

doi: 10.64898/2026.04.27.720696

Figure Lengend Snippet: The complement system is an arm of innate immunity, comprising ∼50 proteins present throughout 8ssues and ac8vated by proteoly8c cleavage. a) Complement ac8va8on occurs via one of three pathways: classical, lec8n, and alterna8ve. The classical pathway is ini8ated when C1q binds targets, ac8va8ng C1r and C1s, which cleave C4 and C2 to form the C3 convertase, C4b2a. The lec8n pathway is triggered by mannose-binding lec8n (MBL), collec8ns, or ficolins recognizing microbial- or altered self-carbohydrates, ac8va8ng MASP-1 and MASP-2 to generate the C3 convertase, C4b2a. The alterna8ve pathway is cons8tu8vely ac8ve through spontaneous C3 hydrolysis, forming the free C3 convertase C3(H2O)Bb, and is amplified on surfaces to form the alterna8ve pathways C3 convertase, C3bBb, enhancing complement ac8va8on. b) The central event in the cascade is cleavage of C3 by the C3 convertases to form C3a and C3b, which ini8ate the major effector responses of the system. c) Opsoniza9on/Phagocytosis : C3b and iC3b deposited on target surfaces promote phagocytosis via CR1, CR3, and CR4. Inflammatory Signaling : The anaphylatoxin pep8des, C3a and C5a, mediate inflammatory signaling through C3aR and C5aR, driving chemotaxis, cytokine produc8on, and ac8va8on of immune cells. Membrane ACack Complex (MAC) : C5b ini8ates the terminal pathway by recrui8ng C6–C9 to form the MAC, lysing target cells. Complement ac8vity is 8ghtly regulated at mul8ple levels (shown in pink boxes): C1 inhibitor (C1-INH) and neuronal pentraxins (NPTXs) restrain classical ini8a8on, C4 binding protein (C4BP), Factor H (FH), and FI regulate C3 convertases and alterna8ve pathway amplifica8on; carboxypep8dase-N (CPN) inac8vates anaphylatoxins; the membrane bound regulators CR1, CD46, CD55, and CD59 prevent deposi8on of ac8vated complement on self-cells; clusterin (CLU) and vitronec8n (VTN) prevent MAC assembly and inser8on. Representa8ve complement components (shown in red text) were quan8fied in α-synuclein pre-formed fibril–injected rats and postmortem PD brains.

Article Snippet: Wells were then washed four times with 200μL of PBS-T and incubated in 50μL of a primary antibody specific to a neo-epitope in cleaved (activated) complement C3 (1:500; Hycult HM2257; RRID:AB_1953566) diluted in 2% BSA for 1 hour at RT with gentle shaking.

Techniques: Binding Assay, Amplification, Chemotaxis Assay, Membrane, Injection

Journal: bioRxiv

Article Title: Complement Dysregulation During the Early Phases of Synucleinopathy

doi: 10.64898/2026.04.27.720696

Figure Lengend Snippet: Rats (n=6-8/sex/group) received intra-striatal injec8ons of α-synuclein (α-syn) preformed fibrils (PFFs) or phosphate buffer saline (PBS) and were sacrificed 2-months post-injec8on. A-b) Representa8ve images of Serine 129 phosphorylated α-syn (pSyn) immunostaining in the substan8a nigra (SN) of PBS ( a ) and α-syn PFF ( b ) injected rats. C-d ) Representa8ve images of MHC-II immunostaining in the SN of PBS ( c ) and α-syn PFF ( d ) injected rats. High magnifica8on images to the right of each panel correspond to the area in the box of respec8ve low magnifica8on images. E-F ) Droplet digital PCR (ddPCR) quan8fica8on of complement component 3 ( C3) expression in the striatum (ST; panel e ) and SN ( f ). Data are C3 normalized to ribosomal potein L13 ( Rpl13 ), analyzed with t-test with Welch’s correc8on. g ) Representa8ve immunoblot of pSyn, α-syn and β-ac8n from the ST. h ) Quan8fica8on of pSyn monomers (∼14-17 kDa), i ) pSyn mul8mers (∼20-50 kDa), j ) total pSyn signal (∼14-50 kDa), and k ) α-syn monomer (∼14-17 kDa) normalized to β-ac8n in the ST. l) Representa8ve immunoblot of pSyn, α-syn and β-ac8n from the SN. m ) Quan8fica8on of pSyn monomers (∼14-17 kDa), n ) pSyn mul8mers (∼20-50 kDa), o ) total pSyn signal (∼14-50 kDa), and p ) α-syn monomer (∼14-17 kDa) normalized to β-ac8n in the SN. q) Representa8ve immunoblot of C3 and β-ac8n from the ipsilateral ST. s ) Representa8ve immunoblot of C3 and β-ac8n from the ipsilateral SN Quan8fica8on of C3 whole molecule (∼190 kDa), C3 α-chain (∼115 kDa), iC3b α-chain (∼75 kDa), and C3c α-chain (∼34kDa) normalized to β-ac8n from the ST ( r ) and SN ( t ), analyzed with t-test with Welch’s correc8on). Scale bars in large images of ( b, d) are 250μm and apply to large images of ( a-d) , while scale bar in the small images of ( b, d) are 50μm and apply to small images of ( a-d) . All data are group means ± standard devia8on expressed a fold change from PBS group.

Article Snippet: Wells were then washed four times with 200μL of PBS-T and incubated in 50μL of a primary antibody specific to a neo-epitope in cleaved (activated) complement C3 (1:500; Hycult HM2257; RRID:AB_1953566) diluted in 2% BSA for 1 hour at RT with gentle shaking.

Techniques: Saline, Immunostaining, Injection, Digital PCR, Expressing, Western Blot

Journal: bioRxiv

Article Title: Complement Dysregulation During the Early Phases of Synucleinopathy

doi: 10.64898/2026.04.27.720696

Figure Lengend Snippet: Male and female rats (n=5-6/group) received intra-striatal injec8ons of α-synuclein (α-syn) preformed fibrils (PFFs) or phosphate buffer saline (PBS) and were sacrificed 2 months post injec8on. a-h ) Representative low magnification immunofluorescent (IF) images of Huc/d (cyan, neuronal marker), serine 129 phosphorylated α-syn (pSyn; green) and complement component 3 (C3; red) and the overlay image in the SNc of PBS ( a-d ) and PFF ( e-h ) injected rats. i-p ) High magnification images corresponding to box in ( d ) and ( h ), for PBS ( i-l ) and PFF injected ( m-p ) rats, respectively. q-r ) Quantification of C3 ( q ) and pSyn ( r ) fluorescence intensity. s ) Regression analysis between pSyn and C3 fluorescence intensity in the SNc of PFF injected rats. t-u ) Quantification of percent area of SNc occupied by C3+ ( t ) and pSyn+ ( u ) staining. v ) Regression analysis between pSyn and C3 percent area staining in the SNc of PFF injected rats. Data expressed as mean fold change (± standard deviation) from PBS controls, analyzed by t-test with Welch’s correction or simple linear regression analyses. w-z ) Representative IF images of pSyn (green; panel w ), the microglial marker, ionized calcium binding adapter molecule 1 (IBA1; cyan; panel x ), C3 (red; panel; y ) and the overlay image ( z ) in the SNc of PFF injected rats. Arrows in ( m-p ) and ( w-z ) indicate areas where C3+ microglia are in direct apposition of neurons containing pSyn+ aggregates. Scale bars in ( h ) is 250μm and applies to ( a-h ), scale bar in ( p ) is 50μm and applies to ( i-p ), scale bar in ( z ) is 50μm and applies to ( w-z )

Article Snippet: Wells were then washed four times with 200μL of PBS-T and incubated in 50μL of a primary antibody specific to a neo-epitope in cleaved (activated) complement C3 (1:500; Hycult HM2257; RRID:AB_1953566) diluted in 2% BSA for 1 hour at RT with gentle shaking.

Techniques: Saline, Marker, Injection, Fluorescence, Staining, Standard Deviation, Binding Assay

Journal: bioRxiv

Article Title: Complement Dysregulation During the Early Phases of Synucleinopathy

doi: 10.64898/2026.04.27.720696

Figure Lengend Snippet: Male and female rats (n=5-6/group) received intra-striatal injec8ons of α-synuclein (α-syn) preformed fibrils (PFFs) or phosphate buffer saline (PBS) and sacrificed 2-months post-injec8on. a-h ) Representative low magnification images of DAPI (blue, nuclear marker), pSyn (green, phospho-Ser129 α-syn) and C3 (red, complement 3) and the overlay in the ipsilateral cortex (Cx) of PBS ( a-d ) and α-syn PFF ( e-h ) injected rats. i-p) High magnification images corresponding to box in ( d ) and ( h ), for PBS ( i-l ) and PFF ( m-p ) injected rats, respectively. q-r ) Quantification of C3 ( q ) and pSyn ( r ) fluorescence intensity in the Cx. s ) Linear regression between pSyn and C3 fluorescence intensity in the Cx of PFF injected rats. t-u) Quantification of percent area of Cx occupied by C3+ ( t ) and pSyn+ ( u ) staining. v ) Linear regression between pSyn and C3 percent area staining in the Cx of PFF injected rats. w-dd ) Representative low magnification images of DAPI (blue), pSyn (green) and C3 (red) and overlay image in the ipsilateral striatum (ST) of PBS ( w-z ) and PFF ( aa-dd ) injected rats. ee-ll) High magnification images corresponding to box in ( z ) and ( dd ), for PBS ( ee-hh ) and PFF ( ii-ll ) injected rats, respectively. mm-nn ) Quantification of C3 ( mm ) and pSyn ( nn ) fluorescence intensity in the ST. oo ) Linear regression between pSyn and C3 fluorescence intensity in the ST of PFF injected rats. pp-qq) Quantification of percent area of ST occupied by C3+ ( pp ) and pSyn+ ( qq ) staining. rr ) Linear regression between pSyn and C3 percent area staining in the ST of PFF injected rats. Data expressed as mean fold change (± standard deviation) from PBS controls (analyzed with unpaired t-test with Welch’s correction or simple linear regression analyses). Scale bars in ( h; dd) are 250μm and apply to ( a-h; w-dd) , respectively. Scale bars in ( p; ll) are 50μm and apply to ( i-p; ee-ll) , respectively.

Article Snippet: Wells were then washed four times with 200μL of PBS-T and incubated in 50μL of a primary antibody specific to a neo-epitope in cleaved (activated) complement C3 (1:500; Hycult HM2257; RRID:AB_1953566) diluted in 2% BSA for 1 hour at RT with gentle shaking.

Techniques: Saline, Marker, Injection, Fluorescence, Staining, Standard Deviation

Journal: Biomolecules

Article Title: Complement Activation May Drive the Pathogenicity of Anti-α6 and Anti-β4 Integrin Antibodies In Vivo

doi: 10.3390/biom16030417

Figure Lengend Snippet: Anti-α6 and anti-β4 integrin IgG fail to fix complement along the BMZ in vitro. Representative indirect immunofluorescence microscopy images of murine buccal mucosa, conjunctiva, and skin biopsies are shown. Following incubation of sections with IgG, fresh hirudin plasma was added as a complement source. Complement activation was assessed using anti-C3 staining. Robust linear C3 deposition (arrows) along the BMZ was observed in normal murine skin treated with IgG against the recombinant NC1 domain of collagen type VII (anti-COL7 IgG, positive control). In contrast, no C3 deposition was detected in tissues treated with anti-α6 integrin IgG or anti-β4 integrin IgG, indicating their inability to activate complement in vitro. Sections incubated with normal rabbit (NR) IgG (negative control) served as negative controls. Nuclei were counterstained with DAPI (blue). Scale bars, 100 μm.

Article Snippet: Subsequently, the sections were stained with FITC-conjugated anti-human complement C3 IgG (dilution 1:100; MP Biomedicals, Eschwege, Germany) for 1 h at 37 °C.

Techniques: In Vitro, Immunofluorescence, Microscopy, Incubation, Clinical Proteomics, Activation Assay, Staining, Recombinant, Positive Control, Negative Control