cd55-fc  (Sino Biological)

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) was immobilized on a Series S Protein G sensor chip (Cytiva) on one flow cell, while a blank flow cell was used as reference.

Techniques: CRISPR, Activation Assay, Transduction, Genome Wide, Staining, Stable Transfection, Expressing, 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) was immobilized on a Series S Protein G sensor chip (Cytiva) on one flow cell, while a blank flow cell was used as reference.

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) was immobilized on a Series S Protein G sensor chip (Cytiva) on one flow cell, while a blank flow cell was used as reference.

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

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

Journal: bioRxiv

Article Title: Plasmodium falciparum exploits CD44 as a co-receptor for erythrocyte invasion

doi: 10.1101/2023.04.12.536503

Figure Lengend Snippet: (A) Mass spectrometry results from affinity purification experiments in which bead-immobilized rCD44-Fc or rFc were incubated with lysate from P. falciparum strain 3D7 schizont-stage parasites. Heat map shows the normalized spectral counts (based on protein size) of the most abundant P. falciparum proteins detected and their enrichment in rCD44-Fc compared to rFc. NaN indicates no counts. The table shows Log probability, raw number of spectra and unique peptides, and coverage of EBA-140 and EBA-175 proteins found in rCD44-Fc lane. (B) Western blot of independent affinity purification experiment in which bead-immobilized rCD44-Fc or rFc were incubated with P. falciparum schizont-stage lysate, followed by immunoblotting for EBA-175 or EBA-140. SN, supernatant. (C) Flow cytometry-based binding assays in which recombinant region RII of EBA-175-His (left) or EBA-140-His (right) were incubated with beads coated with rCD44-Fc, rFc, or no protein. Binding was detected using an anti-His antibody and a fluorescent secondary antibody. (D) Flow cytometry-based binding assays between rCD55-Fc or rFc and region RII of EBA-175-His (left) or EBA-140-His (right). (E) Binding assays of RII EBA-175 (left) or RII EBA-140 (right) with rCD44-Fc in the presence of anti-CD44 monoclonal antibody BRIC 222 or isotype control BRIC 170.

Article Snippet: Protein A Dynabeads (1.5 mg) were incubated with 8 µg rCD44-Fc (R&D systems 3660-CD), rCD44-Fc (SinoBiological 12211-H02H), rCD55-Fc (SinoBiological 10101-H02H), IgG 1 -Fc (R&D systems 110-HG), or no protein in 200 µl antibody binding and washing buffer (Invitrogen kit 10006D) for one hour at RT.

Techniques: Mass Spectrometry, Affinity Purification, Incubation, Western Blot, Flow Cytometry, Binding Assay, Recombinant, Protein Binding, Control