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

Article Title: Epidermal growth factor receptor targeting IgG3 triggers complement-mediated lysis of decay-accelerating factor expressing tumor cells through the alternative pathway amplification loop.

doi: 10.4049/jimmunol.1400329

Figure Lengend Snippet: FIGURE 4. CML triggered by anti–EGFR-IgG3 negatively correlates with CD55 and CD59 expression levels. (A) Surface expression levels of CD46, CD55, and CD59 on analyzed cell lines were quantified by calibrated flow cytometry. Means 6 SEM of at least three independent experiments are presented. (B) Correlations between CD46, CD55, or CD59 and anti–EGFR-IgG3–mediated CDC were calculated for all four cell lines. CDC results at 2 mg/ml Ab concentration were taken from experiments presented in Fig. 2. (C) A431 cells were seeded into 10-cm plates and grown overnight. On the following day, cells were transfected with 50 nM control siRNA or with single siRNAs specific for CD46, CD55, CD59, or with a combination of all three mCRP-specific siRNAs for 72 h. Efficiency of siRNA-induced knockdown was analyzed by direct flow cytometry using fluorochrome-labeled, mCRP- specific Abs (CD46-Pacific blue, CD55-PE, CD59-FITC), or respective control Abs. (D–G) CDC against control siRNA or mCRP-specific, siRNA- transfected A431 cells was analyzed by 3-h [51Cr] release assays in the presence of 25% v/v human serum and anti–EGFR-IgG1 (upper panels), anti– EGFR-IgG3 (lower panels), as well as the respective control Abs at increasing concentrations. (H) Concentration-dependent binding of CD55-Ab (BRIC216, mouse IgG1) to A431 cells was analyzed by indirect immunofluorescence. Results from one representative experiment are presented. (I) CDC triggered by anti–EGFR-IgG1, anti–EGFR-IgG3, or respective control Abs (all at 66.67 nM) against A431 cells in the presence of saturating concentrations of CD55-Ab or a control Ab (both at 66.67 nM) was analyzed by 3-h [51Cr] release assays in the presence of 25% v/v human (Figure legend continues)

Article Snippet: To block complement regulatory activity of CD55, we used mouse anti-human CD55 (66.67 nM, BRIC216, mouse IgG1; Bio-Rad) blocking mAb in CDC experiments at saturating concentrations.

Techniques: Expressing, Cytometry, Concentration Assay, Transfection, Control, Knockdown, Labeling, Binding Assay

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

Article Title: Epidermal growth factor receptor targeting IgG3 triggers complement-mediated lysis of decay-accelerating factor expressing tumor cells through the alternative pathway amplification loop.

doi: 10.4049/jimmunol.1400329

Figure Lengend Snippet: FIGURE 5. CD55 dampens anti–EGFR-IgG3–triggered CML and promotes C1q-dependent induction of AP amplification. BHK-EGFR+ #5 cells were transiently transfected with a control vector or a CD55 vector for 48 h. (A) Cell-surface expression of CD55 was analyzed by direct flow cytometry using PE-conjugated CD55-specific or control Abs. (B–D) The influence of CD55 overexpression on anti–EGFR-IgG3–mediated CDC was investigated by [51Cr] release assays either (B) in an Ab concentration–response curve, (C) in a time-dependent manner, or (D) in serum titration experiments. (E–H) The influence of the alternative complement pathway inhibitor CRIg (E and G), the presence of C1q in serum (F; at 66.67 nM Ab concentration; mean 6 SEM of triplicates), as well as of factor B (H; 13.33 nM Ab concentration, 12.5% v/v factor B–depleted serum, 200 mg/ml factor B), on anti–EGFR-IgG3–mediated CDC was analyzed using either (E and F) control vector–transfected or CD55 vector–transfected BHK-EGFR+ #5 cells or (G and H) DiFi cells (66.67 nM Ab concentration). (I) Deposition of factor Bb on control vector– or CD55 vector–transfected BHK-EGFR+ #5 cells was analyzed by flow cytometry. Relative deposition levels were calculated by equating RFI measured in the absence of Ab with 100%. Results are presented as mean 6 SEM of at least three independent experiments with different blood donors. *p # 0.05 anti–EGFR-IgG3 versus respective control Ab; (B–D, I) #p # 0.05 control vector versus CD55 vector; (E and G) #p # 0.05 without CRIg-Fc versus CRIg-Fc; (H) #p # 0.05 w/o factor B versus with factor B.

Article Snippet: To block complement regulatory activity of CD55, we used mouse anti-human CD55 (66.67 nM, BRIC216, mouse IgG1; Bio-Rad) blocking mAb in CDC experiments at saturating concentrations.

Techniques: Transfection, Control, Plasmid Preparation, Expressing, Cytometry, Over Expression, Concentration Assay, Titration

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

Article Title: Epidermal growth factor receptor targeting IgG3 triggers complement-mediated lysis of decay-accelerating factor expressing tumor cells through the alternative pathway amplification loop.

doi: 10.4049/jimmunol.1400329

Figure Lengend Snippet: FIGURE 6. Overview of complement activation by human anti–EGFR-IgG3 in the context of CD55 expression. On CD55-deficient target cells (left panel), anti–EGFR-IgG3 mediates strong C3b but low C4b deposition and induces assembly of classical and alternative C3 convertases, predominantly resulting in the induction of fast and efficient CDC via the classical pathway of complement activation. In contrast, on CD55-expressing target cells (right panel), CD55 mainly accelerates the decay of low amounts of classical C3 convertases, leading to amplification of the AP and finally to slow and inefficient CDC induction.

Article Snippet: To block complement regulatory activity of CD55, we used mouse anti-human CD55 (66.67 nM, BRIC216, mouse IgG1; Bio-Rad) blocking mAb in CDC experiments at saturating concentrations.

Techniques: Activation Assay, Expressing

Journal: Clinical and experimental immunology

Article Title: miR-132-3p regulates antibody-mediated complement-dependent cytotoxicity in colon cancer cells by directly targeting CD55.

doi: 10.1093/cei/uxac120

Figure Lengend Snippet: Figure 1: CD55 is a direct target of miR-132-3p. (A) The relative expression level of miRNAs in the Ls-174T cell line was detected by RT-qPCR and compared to the miRNAs levels in the LoVo cell line. (B) miR-132-3p expression level in LoVo or Ls-174T cell lines transfected with the miR-132-3p mimics or inhibitor and their respective negative controls. CD55 mRNA (C) and protein (D) expression in the LoVo or Ls-174T cell line transfected with the miR-132-3p mimics or inhibitor and their respective negative controls. (E) Schematic representation of the CD55-3ʹ-UTR (untranslated region) reporter construct and sequence alignment between miR-132-3p and the CD55 3ʹ-UTR wild-type and CD55 3ʹ-UTR mutant sequences. Mutations were generated based on the miR-132-3p-binding sequence of the CD55 3ʹ-UTR as indicated. Dual-luciferase reporter data with the wild-type and mutated 3ʹ-UTR of CD55 are shown after miR-132-3p overexpression. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The membranes were blocked with 5% skim milk in TBS containing 0.05% Tween 20 at room temperature for 1 h. Membranes were incubated overnight with primary antibodies against CD55 (1:400, ABclonal, Woburn, MA, USA) and β-actin (1:8000, ZenBioScience, Chengdu, China) in 5% non-fat milk at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Construct, Sequencing, Mutagenesis, Generated, Binding Assay, Luciferase, Over Expression

Journal: Clinical and experimental immunology

Article Title: miR-132-3p regulates antibody-mediated complement-dependent cytotoxicity in colon cancer cells by directly targeting CD55.

doi: 10.1093/cei/uxac120

Figure Lengend Snippet: Figure 4: miR-132-3p and CD55 expression level in colon tumour tissues and adjacent normal tissues. (A) H&E staining of frozen tissue samples (×100). (B) Representative images of CD55 expression in human normal colon tissues and colon cancer tissues, as determined through IHC. a: Normal colon tissues. b: Highly differentiated colon cancer tissues. c: Moderately differentiated colon cancer tissues. d: Poorly differentiated colon cancer tissues. (C) Expression of CD55 mRNA and miR-132-3p in paired tumour tissues and normal tissues. (D) Correlation analysis of CD55 protein based on IHC and CD55 based on RT-qPCR (left), as well as CD55 protein based on IHC and miR-132-3p based on RT-qPCR. *P < 0.05, ***P < 0.001.

Article Snippet: The membranes were blocked with 5% skim milk in TBS containing 0.05% Tween 20 at room temperature for 1 h. Membranes were incubated overnight with primary antibodies against CD55 (1:400, ABclonal, Woburn, MA, USA) and β-actin (1:8000, ZenBioScience, Chengdu, China) in 5% non-fat milk at 4°C.

Techniques: Expressing, Staining, Quantitative RT-PCR

Journal: iScience

Article Title: CRISPR-Cas9 screening reveals a distinct class of MHC-I binders with precise HLA-peptide recognition

doi: 10.1016/j.isci.2024.110120

Figure Lengend Snippet: Receptor-ligand CRISPR-Cas9 activation screen reveals that CD55 interacts with HLA-C∗07:01-VRIG tetramers (A) Schematic of the receptor ligand CRISPR-Cas9 activation screen. K562 cells transduced with a genome-wide activation library were stained with a pool of three HLA tetramers (HLA-A∗02:01-NLVP, HLA-B∗07:02-TPRV, and HLA-C∗07:01-VRIG), and enriched gRNAs in stained cells were identified using NGS. (B) SigmaFC scores of genes from two replicate screens. SigmaFC scores were calculated using PinAplPy, and top hits are annotated. (C) K562 cells stably expressing dCas9 and transduced with a gRNA upregulating CD55 or a control guide were stained with the HLA-A, -B, -C, or tetramers as in (A) or with HLA-E∗01:01-VMAP tetramers and analyzed by flow cytometry. (D) In vitro co-immunoprecipitation of recombinant CD55-Fc with HLA-A∗02:01-NLVP, HLA-B∗07:02-TPRV, HLA-C∗07:01-VRIG, or HLA-E∗01:01-VMAP tetramers. (E) Three different cell lines (HeLa, PC-3M, or SiHa) that express CD55 endogenously were stained for CD55 (top) or with HLA-C∗07:01-VRIG tetramers (bottom) and analyzed by flow cytometry. (F) HeLa wild-type or HeLa CD55 KO cells were stained with αCD55 or HLA-C∗07:01-VRIG tetramers and analyzed by flow cytometry. All data except (B) represent at least three independent experiments. CRISPRa, CRISPR activation screen; TMs, tetramers; WT, wild-type; KO, knockout. Related to Figure S1 and Table S1 .

Article Snippet: Antibodies used in this study were anti-heparan sulfate chains (AMSBIO, F58-10E4), FITC anti-human CD55 (Biolegend, 311306), anti-human CD55 BRIC110 (ARP, 08-9402-2, targets SCR2 of CD55), anti-human CD55 BRIC216 (Biorad, MCA914T, targets SCR3 of CD55), anti-human CD55 MAB2009 (R&D systems, MAB2009-SP, targets SCR1 of CD55), anti-SDC2 APC (R&D systems, FAB2965A), anti-SDC4 APC (R&D systems, FAB29181A), anti-CD55 APC (Biolegend, #311311), or goat-anti-mouse APC (Biolegend, #405308).

Techniques: CRISPR, Activation Assay, Transduction, Genome Wide, Staining, Stable Transfection, Expressing, Control, Flow Cytometry, In Vitro, Immunoprecipitation, Recombinant, Knock-Out

Journal: iScience

Article Title: CRISPR-Cas9 screening reveals a distinct class of MHC-I binders with precise HLA-peptide recognition

doi: 10.1016/j.isci.2024.110120

Figure Lengend Snippet: Interaction of CD55 with HLA-C∗07:01-VRIG tetramers is allotype and peptide specific (A) HEK293T cells were transfected with a plasmid containing GFP and a truncation mutant of CD55 and analyzed by flow cytometry. GFP+ positive cells were analyzed for staining with HLA-C∗07:01-VRIG. Each mutant removes an additional SCR domain from CD55. Data are represented as mean ± SD. (B) HeLa cells were stained with HLA-C∗07:01-VRIG tetramers after pre-incubation with CD55 blocking antibodies targeting different SCR domains on CD55 and analyzed by flow cytometry. (C) HeLa cells were stained with either HLA-C∗07:01 or HLA-C∗07:02 tetramers loaded with the VRIG peptide and analyzed by flow cytometry. (D) HeLa cells were stained with HLA-C∗07:01 tetramers loaded with different alanine mutants of the VRIGHLYIL peptide and analyzed by flow cytometry. (E) CD55-Fc was immobilized on a Prot-G chip for SPR data using HLA-C∗07:01-VRIG tetramers as analyte to determine interaction on and off rates and K D . Response units were measured with increasing concentrations of HLA-C∗07:01-VRIG tetramers. All data represent at least three independent experiments, except (E), which represents a biological duplicate. FL, full length. Related to Figure S2 , Tables S2 and .

Article Snippet: Antibodies used in this study were anti-heparan sulfate chains (AMSBIO, F58-10E4), FITC anti-human CD55 (Biolegend, 311306), anti-human CD55 BRIC110 (ARP, 08-9402-2, targets SCR2 of CD55), anti-human CD55 BRIC216 (Biorad, MCA914T, targets SCR3 of CD55), anti-human CD55 MAB2009 (R&D systems, MAB2009-SP, targets SCR1 of CD55), anti-SDC2 APC (R&D systems, FAB2965A), anti-SDC4 APC (R&D systems, FAB29181A), anti-CD55 APC (Biolegend, #311311), or goat-anti-mouse APC (Biolegend, #405308).

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Flow Cytometry, Staining, Incubation, Blocking Assay

Journal: iScience

Article Title: CRISPR-Cas9 screening reveals a distinct class of MHC-I binders with precise HLA-peptide recognition

doi: 10.1016/j.isci.2024.110120

Figure Lengend Snippet:

Article Snippet: Antibodies used in this study were anti-heparan sulfate chains (AMSBIO, F58-10E4), FITC anti-human CD55 (Biolegend, 311306), anti-human CD55 BRIC110 (ARP, 08-9402-2, targets SCR2 of CD55), anti-human CD55 BRIC216 (Biorad, MCA914T, targets SCR3 of CD55), anti-human CD55 MAB2009 (R&D systems, MAB2009-SP, targets SCR1 of CD55), anti-SDC2 APC (R&D systems, FAB2965A), anti-SDC4 APC (R&D systems, FAB29181A), anti-CD55 APC (Biolegend, #311311), or goat-anti-mouse APC (Biolegend, #405308).

Techniques: Virus, Recombinant, Blocking Assay, Genome Wide, Activation Assay, CRISPR, Knock-Out, Mutagenesis, Plasmid Preparation, Software, Imaging

Journal: The FASEB Journal

Article Title: MAP‐2:CD55 chimeric construct effectively modulates complement activation

doi: 10.1096/fj.202300571r

Figure Lengend Snippet: FIGURE 1 Expression of the novel construct MAP-2:CD551-4. (A) Graphical representation of MAP-2 and MAP-2:CD551-4. (B–D) Western immunoblotting of the purified protein MAP-2:CD551-4 probed with mAb anti-MASP-2/Map19 (B), mAb anti-CD55 (C), and mAb anti-FLAG tag (D). rMAP-2 and rCD551-4 produced in-house were used as controls. (E) Direct protein stain of the same purified proteins.

Article Snippet: Membranes were blocked with 5% (w/v) skim milk (Sigma- Aldrich) for 30 min and then incubated with 0.5 μg/mL rat monoclonal antibody (mAb) anti- MASP- 2/ Map19, clone 6G12 (Hycult, Uden, The Netherlands), 1 μg/mL mouse mAb anti- CD55, clone 278803 (MAB2009; R&D Systems, Minneapolis, MN, USA), or 2 μg/mL mouse mAb anti- FLAG- tag (clone 18, produced in- house).

Techniques: Expressing, Construct, Western Blot, Purification, FLAG-tag, Produced, Staining

Journal: The FASEB Journal

Article Title: MAP‐2:CD55 chimeric construct effectively modulates complement activation

doi: 10.1096/fj.202300571r

Figure Lengend Snippet: FIGURE 2 Structural characterization of MAP-2:CD551-4. (A) MAP-2:CD551-4 elution profile from size exclusion chromatography exposed to 2 mM of calcium, 10 mM EDTA, or 10 mM EGTA. Vertical lines represent the molecular weight of the molecular marker, represented in kDa. (B–D) Western immunoblotting of the elution fractions 3–16 of MAP-2 from SEC exposed to 2.5 mM of calcium (B), 10 mM EDTA (C), or 10 mM EGTA (D). MAb anti MASP-2/Map19 clone 6G12 was used to detect MAP-2:CD551-4. Representative blots out of 3 independent replicates are shown. (E and F) Thermal stability of MAP-2, CD55, and MAP-2:CD551-4 exposed to 2 mM calcium or 10 mM EGTA shown as the ratio of the intrinsic fluorescence at 350 and 330 nm (E) and the right the first derivative of the ratio (F). Thermal stability was performed in triplicates.

Article Snippet: Membranes were blocked with 5% (w/v) skim milk (Sigma- Aldrich) for 30 min and then incubated with 0.5 μg/mL rat monoclonal antibody (mAb) anti- MASP- 2/ Map19, clone 6G12 (Hycult, Uden, The Netherlands), 1 μg/mL mouse mAb anti- CD55, clone 278803 (MAB2009; R&D Systems, Minneapolis, MN, USA), or 2 μg/mL mouse mAb anti- FLAG- tag (clone 18, produced in- house).

Techniques: Size-exclusion Chromatography, Molecular Weight, Marker, Western Blot, Fluorescence

Journal: Signal Transduction and Targeted Therapy

Article Title: Non-small cell lung cancers (NSCLCs) oncolysis using coxsackievirus B5 and synergistic DNA-damage response inhibitors

doi: 10.1038/s41392-023-01603-4

Figure Lengend Snippet: Expression profile of CAR and DAF on normal lung and cancer cells and its correlation with CVB5-mediated cytotoxicity. a Expression of CAR and DAF on normal lung cells (MRC-5, WI-38, and HFL) and NSCLC cells analyzed by western blot. Gray value ratios to the first lane of CAR were shown. b , c Normal lung cells infected with CV-B5/F ( b) or CV-B5/JS417 ( c) at 100 MOI and analyzed at 48 h for cell viability by CCK8 assay ( n = 3). Each value represents the mean ± deviation. d Expression of CAR and DAF on wild-type or CAR over-expressing mouse Lewis lung cancer (LLC; LLC-CAR) and colorectal carcinoma (CT26.WT; CT26.WT-CAR). e LLC, LLC-CAR, CT26.WT, CT26.WT-CAR infected with CV-B5/F at 10 MOI was analyzed at 48 h for cell viability by CCK8 assay ( n = 3). Each value represents the mean ± standard deviation. f – i LLC ( f , g) or LLC-CAR ( h , i) were subcutaneously injected into the axillia of C57BL/6 mice. Each mouse received 5 doses of CV-B5/F or with MEM intratumorally. Tumor were measured every day and anatomized ultimately ( n = 3). j NCI-H1299 infected with CV-B5/F (MOI = 0.01) were analyzed at different time points. Each cellular lysate obtained was subjected to immunoblot analysis. Full-length PARP (116 kDa), cleaved-PARP (85 kDa), full-length caspase 3 (35 kDa) and cleaved-caspase 3 (17/19 kDa) were shown. hpi, hours post infection. k NCI-H1299 pretreated with 100 µM Z-VAD-FMK or MOCK and incubated with MEM or CV-B5/F at 0.01 MOI were subjected to immunoblot analysis. Full-length PARP (116 kDa), cleaved-PARP (85 kDa), and cleaved-caspase 3 (17/19 kDa) were shown. l NCI-1299, NCI-H460, and MRC-5 were infected with 1 MOI CV-B5/F for 24 h. Apoptotic population was represented as Annexin V + /7-AAD - or Annexin V +/ 7-AAD + cells. m NCI-H1299 infected with CV-B5/F (MOI = 0.01) were analyzed at 0, 12, 24, and 48 h. p62 (62 kDa) was detected for the whole cell lysis (WCL). n NCI-H1299 was pretreated with 100 μM CQ for 2 h, and then treated with 0.01 MOI CV-B5/F for 24 h. LC3B (14/16 kDa) was detected for the WCL. o, p NCI-H1299 was transfected with mcherry-GFP-LC3B for 24 h, and then treated with 0.01 MOI CV-B5/F with or without 100 μM CQ (10 μM Rapamycin as positive control). Autophagosomes display both GFP and mCherry fluorescence (yellow-green), whereas autolysosomes display only mCherry fluorescence (red) because GFP is denatured by the acidity of the lysosome ( o) . Number of autophagosomes and autolysosomes were enumerated for at least 20 cells ( n = 20, p ). One-way ANOVA was used to analyze the data. *** P < 0.001, **** P < 0.0001, ns, not significant. Scale bars, 10 μm

Article Snippet: Antibodies to the following proteins were used in this study: β-actin (1: 1000, 4970, Cell Signaling Technology), GAPDH (1: 5000, ab8245, Abcam), DAF (1: 1000, 31759, Cell Signaling Technology), CAR (1: 1000, 16984, Cell Signaling Technology), DAF (1: 2000, sc-51733, Santa Cruz Biotechnology), PARP (1: 1000, 9532, Cell Signaling Technology), Caspase 3 (1: 2000, 9662, Cell Signaling Technology), Cleaved-caspase 3 (1: 2000, 9664, Cell Signaling Technology), LC3B (1: 2000, L7543, Sigma), p62 (1: 2000, ab109012, Abcam), DNA-PK (1: 1000, 38186, Cell Signaling Technology), p-DNA-PK (1: 1000, 68716, Cell Signaling Technology), ATM (1: 1000, 2873, Cell Signaling Technology), p-ATM (1: 1000, ab81292, Abcam), H2AX (1: 2000, ab229914, Abcam), p-H2AX (1: 1000, 80312, Cell Signaling Technology), Akt (1: 1000, 4685, Cell Signaling Technology), p-Akt (Ser473, 1: 1000, 4060, Cell Signaling Technology), p-Akt (Thr308, 1: 1000, 13038, Cell Signaling Technology), mTOR (1: 1000, 2983, Cell Signaling Technology), p-mTOR (1: 1000, 5536, Cell Signaling Technology), α/β-tubulin (1: 1000, 2148, Cell Signaling Technology), Bad (1: 1000, 9292, Cell Signaling Technology), p-Bad (Ser136, 1: 1000, 4366, Cell Signaling Technology), p-Bad (Ser112,1: 1000, 5284, Cell Signaling Technology), Bax (1: 1000, 5023, Cell Signaling Technology), Bcl-2 (1: 1000, 3498, Cell Signaling Technology), Bcl-xL (1: 1000, 2764, Cell Signaling Technology), AIF (1: 1000, 5318, Cell Signaling Technology), JNK (1: 1000, 9252, Cell Signaling Technology), p-JNK (1: 1000, 4668, Cell Signaling Technology), Caspase 12 (1: 2000, ab62484, Abcam), CHOP (1: 1000, ab11419, Abcam), PERK (1: 1000, 5683, Cell Signaling Technology), p-PERK (1: 1000, 3179, Cell Signaling Technology), IRE1 (1: 2000, ab96481, Abcam), p-IRE1 (1: 2000, ab48187, Abcam), ATF6 (1: 2000, ab227830, Abcam), eIF2α (1: 2000, 5324, Cell Signaling Technology), p-eIF2α (1: 2000, 3398, Cell Signaling Technology), ATF4 (1: 2000, ab184909, Abcam), GADD34 (1: 1000, 41222, Cell Signaling Technology), IRF9 (1: 1000, 76684, Cell Signaling Technology), p-STAT1 (1: 1000, 9167, Cell Signaling Technology), STING (1: 1000, 13647, Cell Signaling Technology), FAM134B (1: 1000, 83414, Cell Signaling Technology), cGAS (1: 1000, 79978, Cell Signaling Technology), Lamin B1 (1: 1000, ab194109, Abcam), HMGB1 (1: 1000, 6893, Cell Signaling Technology), GSDME (1: 1000, 19453, Cell Signaling Technology), Bip (1: 1000, 3177, Cell Signaling Technology), p53 (1: 2000, ab26, Abcam), p-p53 (Ser15, 1: 2000, ab223868, Abcam), p21 (1: 1000, 2947, Cell Signaling Technology), RIG-I (1:1000, 3743, Cell Signaling Technology), STING (1:500, A3575, Abclonal), V5 (1:1000, 13202, Cell Signaling Technology), GFP (1:1000, ab1218, Abcam).

Techniques: Expressing, Western Blot, Infection, CCK-8 Assay, Standard Deviation, Injection, Incubation, Lysis, Transfection, Positive Control, Fluorescence

Journal: The Journal of Experimental Medicine

Article Title: Loss of decay-accelerating factor triggers podocyte injury and glomerulosclerosis

doi: 10.1084/jem.20191699

Figure Lengend Snippet: ADR induces PLAD-dependent cleavage of DAF. (A and B) Representative images (A) and distribution of DAF expression (B) quantified in hiPod exposed to vehicle, ADR (0.3 µg/ml), PLADi (1 µM), or ADR + PLADi (0.3 µg/ml in 1 µM) for 24 h. DAF IF signal was normalized to actin expression pixel by pixel, and the MFI for each cell was computed. Results are representative of two independent experiments with similar results. (C and D) Representative blots (C) and densitometric analysis (D) of PLAD expression in hiPod cell lysates previously exposed to vehicle or ADR for 24 h. (E and F) Representative blots (E) and densitometric analysis (F) of DAF in the supernatants of hiPod exposed to ADR for 24 h with or without PLADi (WB). (G) Representative blot of DAF in the urine from BALB/c male mice at 2 wk after treatment with vehicle or ADR compared with recombinant mouse DAF (rDAF). In each group, we pooled and concentrated urine samples from eight mice (see Materials and methods). All experimental data were verified in at least three independent experiments. *P < 0.05; n.s., not significant. Scale bars: 50 µm. Error bars are SEM.

Article Snippet: The urine from mouse and human samples was separated on 4–20% precast Protean TGX gels (Bio-Rad Laboratories) with recombinant mouse (55 μg/ml, 5490-CD-050; R&D Systems) or human (15 μg/ml, SRP6437; Sigma-Aldrich) CD55 protein, respectively.

Techniques: Expressing, Recombinant

Journal: The Journal of Experimental Medicine

Article Title: Loss of decay-accelerating factor triggers podocyte injury and glomerulosclerosis

doi: 10.1084/jem.20191699

Figure Lengend Snippet: FSGS in humans is associated with DAF down-regulation and complement activation. (A–D) C3 (A), C3aR (B), C5aR (C), and DAF mRNA (D) expression in glomeruli of human biopsy specimens with pathological diagnosis of FSGS or diabetic kidney disease compared with normal kidneys. Data are from previously published microarray studies by and were subjected to further analysis using Nephroseq. (E–H) Representative renal staining and data quantification for C3d (IF; E and F) and DAF (immunohistochemistry; G and H) in patients with FSGS ( n = 18) and in kidneys from healthy renal donors ( n = 10). (I) Correlation between protein and C3a in urine samples from 27 patients with FSGS taken at the time of kidney biopsy (before therapy). (J and K) Differences in proteinuria (J) and urinary C3a (K) measured before versus 3–6 mo after steroid therapy in a subset of 13 patients with FSGS. (L) Correlation between the change in proteinuria and change in urinary C3a before and after therapy for each of the same 13 patients. (M) Representative blot of DAF in the urine from healthy control individuals and patients with FSGS compared with recombinant human DAF (rDAF). In each group, we pooled and concentrated urine samples from five and five subjects, respectively (see Materials and methods). *P ≤ 0.05. Scale bars: 25 μm. Error bars are SEM.

Article Snippet: The urine from mouse and human samples was separated on 4–20% precast Protean TGX gels (Bio-Rad Laboratories) with recombinant mouse (55 μg/ml, 5490-CD-050; R&D Systems) or human (15 μg/ml, SRP6437; Sigma-Aldrich) CD55 protein, respectively.

Techniques: Activation Assay, Expressing, Biomarker Discovery, Microarray, Staining, Immunohistochemistry, Control, Recombinant