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: bioRxiv

Article Title: The Sez6 family inhibits complement at the level of the C3 convertase

doi: 10.1101/2020.09.11.292623

Figure Lengend Snippet: A) Sez6, Sez6L, and Sez6L2 are expressed by principal (excitatory, pyramidal) neurons of the mouse hippocampus at much higher levels than other known complement regulators (namely Crry, C4BP, CFH, C1-INH, DAF, and MCP). Expression data was obtained from Hipposeq: a comprehensive RNA-Seq database of gene expression in hippocampal principal neurons ( http://hipposeq.janelia.org ; ). The RNA samples used in this database were isolated from mouse hippocampal principal neurons micro-dissected from the CA1, CA3, or Dentate Gyrus (DG) cell layers of the hippocampus at Postnatal Day 25-32. Differential gene expression is shown in the heatmap with the relative units of FPKM (Fragments Per Kilobase of Exon Per Million Reads Mapped.) B) Brain sections from adult WT mice or Sez6 triple knockout mice (TKO) were immuno-stained for Sez6L2 (green) and DAPI and imaged in the CA1 region of the hippocampus. Scale Bar= 27μm. High density Sez6L2 staining occurs around cell bodies in the pyramidal layer, but significant Sez6L2 is also found in the stratum radiatum and stratum oriens. C) Higher magnification images of sections immuno-stained for Sez6L2 (green) and the postsynaptic protein, Homer1 (red), shows Sez6L2 is found near or co-localized with synapses in the stratum radiatum. Scale bar = 1.8μm.

Article Snippet: Alternatively cells were transfected with a GFP expression plasmid or co-transfected with GFP and M-SEZ6L2 or HIS-tagged DAF (H-DAF; Sinobiological HG10101-NH; modified from NCBI RefSeq NM 000574.3 ).

Techniques: Expressing, RNA Sequencing Assay, Isolation, Triple Knockout, Staining

Journal: bioRxiv

Article Title: The Sez6 family inhibits complement at the level of the C3 convertase

doi: 10.1101/2020.09.11.292623

Figure Lengend Snippet: A) Purified Sez6L2-MH is shown by a coomassie stained gel and by western blot with anti-Sez6L2 and anti-Myc antibodies. Lanes with the coomassie stain are from the same gel. B) Schematic of Sez6L2 and Sez6L2-MH domain structures. CCP=Domain abundant in c omplement c ontrol p roteins. CCP domains are also known as SUSHI repeats or short complement-like repeat (SCR) CUB= Domains named after complement C 1r/C1s, u EGF, and B MP1 TM=Transmembrane region. Sez6L2-MH was made by replacing the transmembrane and cytoplasmic tail domains with a tandem Myc, 6xHis tag. C) and D) Classical pathway hemolysis assay. Antibody-coated sheep erythrocytes were exposed to human serum pre-incubated with purified Sez6L2-MH, C1-INH, FH, C4BP, H-DAF, or BSA. After 30 mins, the percent of cell lysis was measured by spectrophotometry (A415). C1-INH, FH, C4BP, and H-DAF are known complement inhibitors and were used as positive controls. BSA was used as a negative control for comparison. H-DAF is His-tagged DAF. 1-way ANOVA (P <0.0001; F(6,14)=314.4). N=3 (1 experiment with 3 replicates; Representative of 4 independent experiments with Sez6L2 and buffer and 2-3 experiments with each control protein). D) Supernatants from classical pathway hemolysis assays were further analyzed for C3 cleavage products by western blot. Supernatants in D are from same experiment shown in C. Sez6L2-MH increases the amount of the C3dg cleavage product similar to other complement regulators that work at the level of the C3 convertase. Sez6L2-MH runs just above the C3α band of C3. When large amounts of Sez6L2 are present it can cause C3α to run lower on the gel and sometimes mildly interferes with antibody binding to C3α. The membrane blotted for C3 was also stained with ponceau S to reveal total protein and shows the presence of the purified proteins in each sample. E) Schematic of C3 cleavage products. The C3d region recognized by our C3 antibody is highlighted in dark grey. F) Alternative pathway hemolysis assay. Rabbit erythrocytes were exposed to human serum pre-incubated with Sez6L2-MH, complement regulators, or BSA in presence of 12.5mM MgEGTA to block the classical pathway. Then the percent of cell lysis was measured by spectrophotometry (A415). 1-way ANOVA (P =0.0122; F(4,7)=7.298). Results are the mean of three independent experiments which tested 4 independent Sez6L2-MH preps multiple times. N=3 for buffer and Sez6L2-MH; N=2 for FH, H-DAF, and BSA. For all graphs * = p <0.05; ** = p<0.01

Article Snippet: Alternatively cells were transfected with a GFP expression plasmid or co-transfected with GFP and M-SEZ6L2 or HIS-tagged DAF (H-DAF; Sinobiological HG10101-NH; modified from NCBI RefSeq NM 000574.3 ).

Techniques: Purification, Staining, Western Blot, Hemolysis Assay, Incubation, Lysis, Spectrophotometry, Negative Control, Binding Assay, Blocking Assay

Journal: bioRxiv

Article Title: The Sez6 family inhibits complement at the level of the C3 convertase

doi: 10.1101/2020.09.11.292623

Figure Lengend Snippet: A) Schematic of Factor I and cofactor cleavage of C3b and iC3b. B) Factor I cleavage assay of C3b. C3b and Factor I (FI) were incubated alone, with Factor H (FH), or C4BP, or with concentrations of Sez6L2-MH ranging from 1 to 10 μg/mL for 2 hours at 37°C. Then samples were analyzed by western blot using antibodies that recognize C3d, a region within the C3α chain (and highlighted by the black rectangle in the schematics in (A)). Coomassie stained gels are also shown. FH and C4BP are known co-factors of FI towards C3b and served as positive controls. Incubation of C3b and FI with Sez6L2-MH also generated the C3 cleavage products α’1 and α’2 showing Sez6L2-MH is a cofactor for Factor I cleavage of C3b. C) Schematic of Factor I + cofactor cleavage of C4b. D) C4b and Factor I (FI) were incubated alone, with FH, C4BP, or with concentrations of Sez6L2-MH ranging from 1 to 10 μg/mL for 2 hours at 37°C. Then samples were analyzed by western blot using a C4 polyclonal antibody or coomassie stained gels. C4 components recognized by the C4 antibody are colored black in the schematic in C. C4BP is a known cofactor of FI for C4b cleavage and served as a positive control. Incubation of C4b and FI with Sez6L2-MH did not result in the appearance of C4b cleavage products.

Article Snippet: Alternatively cells were transfected with a GFP expression plasmid or co-transfected with GFP and M-SEZ6L2 or HIS-tagged DAF (H-DAF; Sinobiological HG10101-NH; modified from NCBI RefSeq NM 000574.3 ).

Techniques: Cleavage Assay, Incubation, Western Blot, Staining, Generated, Positive Control

Journal: bioRxiv

Article Title: The Sez6 family inhibits complement at the level of the C3 convertase

doi: 10.1101/2020.09.11.292623

Figure Lengend Snippet: Factor I cleavage assay of C3b (A) or C4b (B). C3b or C4b and Factor I (FI) were incubated alone, with Factor H (FH, 1ug), C4BP (1ug), or Sez6L2-MH (1ug or 5ug) for 4 or 8 hours at 37°C. Then samples were analyzed by coomassie stained gels. Sez6L2-MH aids significant factor I cleavage of C3b but not C4b at 4 and 8 hours resulting in appearance of the C3b cleavage products, C3α’1 and C3α’2, but not the C4b cleavage products, C4d and C4α3.

Article Snippet: Alternatively cells were transfected with a GFP expression plasmid or co-transfected with GFP and M-SEZ6L2 or HIS-tagged DAF (H-DAF; Sinobiological HG10101-NH; modified from NCBI RefSeq NM 000574.3 ).

Techniques: Cleavage Assay, Incubation, Staining

Journal: bioRxiv

Article Title: The Sez6 family inhibits complement at the level of the C3 convertase

doi: 10.1101/2020.09.11.292623

Figure Lengend Snippet: A and B) Alternative C3 convertase assay: A 96 well plate coated with C3b was incubated with Factor B and Factor D to form the C3 convertase C3bBb, then incubated with Sez6L2-MH or FH at concentrations ranging from 0 to 500 μg/mL to assess their decay accelerating activity. Factor B remaining bound to the plate (as C3bBb) was detected using an anti-Factor B antibody ELISA in (A) and Bb released into the supernatant is shown via western blot (B). C) Classical C3 convertase assay: A plate coated with C4b was incubated with C2 and C1s-enzyme to form the classical/lectin pathway C3 convertase C4b2a, then incubated with Sez6L2-MH or H-DAF at concentrations ranging from 0 to 500 μg/mL to assess their decay accelerating activity. C2 remaining bound to the plate (presumably as C4b2a) was detected using an anti-C2 antibody ELISA. For A and C ELISAs: N=3 (1 experiment with 3 replicates; representative of 2-3 independent experiments). Statistics: 1-way ANOVAS with Holms-Sidak multiple comparison’s tests were performed for each complement inhibitor with comparisons to 0 ug/mL controls. * p<0.05.

Article Snippet: Alternatively cells were transfected with a GFP expression plasmid or co-transfected with GFP and M-SEZ6L2 or HIS-tagged DAF (H-DAF; Sinobiological HG10101-NH; modified from NCBI RefSeq NM 000574.3 ).

Techniques: Convertase Assay, Incubation, Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: bioRxiv

Article Title: The Sez6 family inhibits complement at the level of the C3 convertase

doi: 10.1101/2020.09.11.292623

Figure Lengend Snippet: A-B) Sez6L2 inhibits C3 deposition at a range of serum concentrations. CHO cells were transfected with plasmids for GFP alone or with Myc-tagged Sez6L2 (M-Sez6L2) or His-tagged DAF (H-DAF). CHO cells were coated with antibodies and exposed to 0-20% C5 depleted human serum for 1 hour and then labeled with anti-C3b/C3c antibodies and analyzed by flow cytometry. One experiment is shown that is representative of two independent experiments. B) C3 deposition on GFP transfected cells with or without M-Sez6L2 or H-DAF at 15% serum. ANOVA (P=0.0016; F(2,6)=22.51). N=3; 1 experiment with 3 replicates (representative of 3+ independent experiments). C) Schematic of Sez6L2, Sez6, and Sez6L protein domain structures. D-I) CHO cells were transfected with the indicated Myc-tagged cDNAs and processed as outlined in A with 15% C5 depleted serum, except that an anti-Myc antibody was used in place of GFP to identify transfected and expressing CHO cells. D) 5% Contour plots of C3 versus Myc fluorescence (top layer) and C3 fluorescence histograms (bottom layer) of the same samples normalized to mode and compared to baseline cells not exposed to serum. For Contour plots, boxed regions highlight cells designated as myc positive (top box) and myc negative (lower box) populations. For C3 histograms, dark grey, solid line population = Myc positive cells; Light grey, dotted line population= Myc negative cells; White, dashed grey line population = baseline. Representative of 4+ independent experiments. E) Quantification of the average median C3 fluorescence intensity from myc positive and myc negative cells within each sample. Statistics = t-tests. N=3 (1 experiment with 3 replicates; Representative of 4+ independent experiments). F) Average median C3 fluorescence intensities after normalization to the myc negative cells from each experimental group. ANOVA between Myc+ cell populations (p<0.001; F(4, 15)=64.53). Sez6L2 inhibits C3 deposition at a level comparable to positive control MCP. Sez6 is a stronger complement inhibitor than Sez6L2 and Sez6L is a weaker inhibitor. F) Average median Myc fluorescence intensity from myc positive cells ANOVA (p<0.001; F(4, 15)=36.79). G) Average % of Myc positive cells in each experimental group (ANOVA, p=0.115; F(4, 15)=2.224). For sections F-H, N=4 (4 independent experiments). I) Sez6 blocks complement deposition more efficiently than Sez6L2 and Sez6L even when comparing similar levels of myc surface expression. Average C3 median fluorescence intensity normalized to internal myc negative populations for M-Sez6, M-Sez6L2, and M-Sez6L samples shown relative to the myc median fluorescence intensity. N=3 (1 experiment with 3 replicates, Representative of 3 independent experiments). For all graphs * = p <0.05; ** = p<0.01; # = p<0.001 for all Myc+ groups compared to M-CR2.

Article Snippet: Alternatively cells were transfected with a GFP expression plasmid or co-transfected with GFP and M-SEZ6L2 or HIS-tagged DAF (H-DAF; Sinobiological HG10101-NH; modified from NCBI RefSeq NM 000574.3 ).

Techniques: Transfection, Labeling, Flow Cytometry, Expressing, Fluorescence, Positive Control

Journal: bioRxiv

Article Title: The Sez6 family inhibits complement at the level of the C3 convertase

doi: 10.1101/2020.09.11.292623

Figure Lengend Snippet: Sez6 family FASTAs sequences were uploaded into the web-based program provided by Ojha et al ( http://coredo.nccs.res.in/meme-5.0.3/CoReDo/home.html ). Classical model complement regulatory proteins (CRPs) have motifs in the order of M5, M3, M1, M2, and either M4 or M1 spaced across 3 consecutive CCP domains. Sez6L2 has this same pattern, but Sez6 and Sez6L do not.

Article Snippet: Alternatively cells were transfected with a GFP expression plasmid or co-transfected with GFP and M-SEZ6L2 or HIS-tagged DAF (H-DAF; Sinobiological HG10101-NH; modified from NCBI RefSeq NM 000574.3 ).

Techniques:

Journal: Cancers

Article Title: Marker Identification of the Grade of Dysplasia of Intraductal Papillary Mucinous Neoplasm in Pancreatic Cyst Fluid by Quantitative Proteomic Profiling

doi: 10.3390/cancers12092383

Figure Lengend Snippet: Experimental workflow. The overall experimental workflow comprises 3 sections: (1) Preparation of 30 individual samples, (2) peptide library construction, and (3) validation by ELISA. The cohort for label-free quantification included 30 pancreatic cyst fluid samples (10 LGD, 5 HGD, 5 invasive IPMN, 5 MCN, and 5 SCN). After mucus removal by sonication, the samples were centrifuged to isolate supernatant. Pooled cyst fluid (comprising equal amounts of 30 individual samples), secreted proteins from PANC1, Mia Paca-2, BxPC3, and pooled cell lysates from the 3 cell lines were compiled to generate a peptide library. All samples were precipitated using cold acetone to extract the protein. After FASP digestion, only the samples that were used to construct the peptide library were subjected to high-pH reverse-phase peptide fractionation. All peptides were analyzed on a Q Exactive mass spectrometer. CD55, one of the potential markers of IPMN dysplasia, was validated by ELISA. PPT, precipitation; FASP, filter-aided sample preparation; LGD, low-grade dysplasia; HGD, high-grade dysplasia; MCN, mucinous cystic neoplasm; SCN, serous cystic neoplasm; SPNT, supernatant.

Article Snippet: CD55 protein was measured using a commercial quantikine ELISA kit (CSB-E05121h, CUSABIO, China) per the manufacturer’s instructions.

Techniques: Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Quantitative Proteomics, Sonication, Construct, Peptide Fractionation, Mass Spectrometry, Sample Prep

Journal: Cancers

Article Title: Marker Identification of the Grade of Dysplasia of Intraductal Papillary Mucinous Neoplasm in Pancreatic Cyst Fluid by Quantitative Proteomic Profiling

doi: 10.3390/cancers12092383

Figure Lengend Snippet: Volcano plots of differentially expressed proteins in three comparison groups. Student’s t -test ( p < 0.05) was conducted for comparisons 1 (low-grade dysplasia (LGD) versus high-grade dysplasia (HGD)) ( A ), 2 (HGD versus invasive IPMN) ( B ), and 3 (LGD versus invasive intraductal papillary mucinous neoplasm (IPMN)) ( C ) to discover differentially expressed proteins (DEPs). The DEPs that were significantly expressed in each comparison group are indicated as colored dots (red: upregulated DEPs, blue: downregulated DEPs). Several marker candidates, including the validation target CD55, are highlighted in each comparison. DEP, differentially expressed protein.

Article Snippet: CD55 protein was measured using a commercial quantikine ELISA kit (CSB-E05121h, CUSABIO, China) per the manufacturer’s instructions.

Techniques: Comparison, Marker, Biomarker Discovery

Journal: Cancers

Article Title: Marker Identification of the Grade of Dysplasia of Intraductal Papillary Mucinous Neoplasm in Pancreatic Cyst Fluid by Quantitative Proteomic Profiling

doi: 10.3390/cancers12092383

Figure Lengend Snippet: Six of the 19 potential markers that were differentially expressed in accordance with the degree of IPMN malignancy. DEFA3 ( A ), MUC13 ( B ), CD55 ( C ), CPS1 ( D ), AMY2A ( E ), and CPB1 ( F ) were differentially expressed, in accordance with the histological grades of IPMN. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; NS, not available.

Article Snippet: CD55 protein was measured using a commercial quantikine ELISA kit (CSB-E05121h, CUSABIO, China) per the manufacturer’s instructions.

Techniques:

Journal: Cancers

Article Title: Marker Identification of the Grade of Dysplasia of Intraductal Papillary Mucinous Neoplasm in Pancreatic Cyst Fluid by Quantitative Proteomic Profiling

doi: 10.3390/cancers12092383

Figure Lengend Snippet: The dynamic range of protein fold-changes in comparisons 1 and 3 and the results of upstream regulator analysis in Ingenuity Pathway Analysis (IPA). The dynamic range with marked fold-changes of the 19 final marker candidates in comparisons 1 (LGD versus HGD) ( A ) and 3 (LGD versus invasive IPMN) ( B ). The red and blue dots indicate the log2-transformed fold-changes of the 7 upregulated and 12 downregulated proteins. The yellow dot represents the log2-transformed fold-change of CEA protein. ( C ) Five potential markers (MUC13, CD55, CPS1, SOD2, and LEFTY1) and their upstream regulators are connected by dotted lines. The molecular types of each upstream regulator are shown in parentheses. Each marker candidate is listed in order of decreasing fold-change, whereas the upstream regulators are listed in order of increasing p-values. LGD, low-grade dysplasia; HGD, high-grade dysplasia; INV, invasive IPMN.

Article Snippet: CD55 protein was measured using a commercial quantikine ELISA kit (CSB-E05121h, CUSABIO, China) per the manufacturer’s instructions.

Techniques: Marker, Transformation Assay

Journal: Cancers

Article Title: Marker Identification of the Grade of Dysplasia of Intraductal Papillary Mucinous Neoplasm in Pancreatic Cyst Fluid by Quantitative Proteomic Profiling

doi: 10.3390/cancers12092383

Figure Lengend Snippet: Validation of CD55 as a potential biomarker target by ELISA. ELISA was performed with CD55-specific antibodies to evaluate the validity of CD55 as a potential biomarker. The concentration patterns of CD55 by ELISA were generally consistent with the LFQ intensities. The CD55 concentrations are indicated according to two types of IPMN classification. The median CD55 concentrations were highest in invasive IPMN ( A ) and high-risk IPMN ( B ). LGD, low-grade dysplasia; HGD, high-grade dysplasia; INV, invasive IPMN; MCN, mucinous cystic neoplasm; SCN, serous cystic neoplasm; *, p < 0.05; **, p < 0.01; NS, not available.

Article Snippet: CD55 protein was measured using a commercial quantikine ELISA kit (CSB-E05121h, CUSABIO, China) per the manufacturer’s instructions.

Techniques: Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Concentration 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: 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: CD55-Fc , Sino Biological , Cat: 10101-H02H.

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: CD55-Fc , Sino Biological , Cat: 10101-H02H.

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: CD55-Fc , Sino Biological , Cat: 10101-H02H.

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