Journal: Cancer Immunology Research

Article Title: MicroRNAs Affect Complement Regulator Expression and Mitochondrial Activity to Modulate Cell Resistance to Complement-Dependent Cytotoxicity

doi: 10.1158/2326-6066.cir-18-0818

Figure Lengend Snippet: Figure 6. Modulation of the membrane complement regulatory proteins by microRNAs. The expression of CD46 (A), CD55 (B), and CD59 (C) in lysates of K562 cells transfected with microRNA inhibitors or with control, determined by the proteomic analysis described in Materials and Methods, is shown as the mean SD of log2 of intensity value units of five independent experiments. , P < 0.05; , P < 0.01, relative to control (Student t test). NS, not significant. D, K562 cells were transfected with miR-616 inhibitor plasmid or with control (C) plasmid as a negative control. After 48 hours, the cells were labeled with anti-CD46, anti-CD55, or anti-CD59 and then with fluorescently labeled secondary antibody. Cells were then analyzed by flow cytometry, and the mean fluorescence intensity (MFI) values, representative of three independent experiments, were determined. The expression of each regulator in anti–miR-616–treated cells was normalized to their levels in control cells (set as 100). , P < 0.05; , P < 0.01, relative to control (Student t test). E–G, K562 cells were transfected with a miR-150 expression plasmid or a control plasmid. After 24 hours, the cells were labeled with mouse anti-CD46 (E), anti-CD55 (F), or anti-CD59 (G) and fluorescently labeled secondary antibody. Cells were then analyzed by flow cytometry, and MFI values, representative of three independent experiments, were determined. , P < 0.05, relative to control (Student t test).

Article Snippet: Mouse monoclonal antihuman CD46 (clone MEM-258), anti-human CD55 (clone 67), and anti-human CD59 (clone MEM-43) antibodies were purchased from AbD Serotec.

Techniques: Membrane, Expressing, Transfection, Control, Plasmid Preparation, Negative Control, Labeling, Cytometry

Journal: Molecular Medicine Reports

Article Title: The effect of anti-HLA class I antibodies on the immunological properties of human glomerular endothelial cells and their modification by mTOR inhibition or GCN2 kinase activation

doi: 10.3892/mmr.2021.11994

Figure Lengend Snippet: Effect of anti-HLAI on ICAM-1, HLA-DR, CD46 and CD59, and the impact of halofuginone or everolimus treatment. (A) Representative experiment for each of the evaluated factors. (B) Cumulative results are presented. Anti-HLAI antibodies upregulated ICAM-1, HLA-DR, CD46 and CD59. Halofuginone or everolimus treatment decreased ICAM-1. Data are presented as the mean ± SEM. *P<0.05 vs. control cells, # P<0.05 vs. anti-HLAI-treated cells, ^ P<0.05 vs. anti-HLAI-treated cells administered halofuginone, + P<0.05 vs. anti-HLAI-treated cells administered everolimus and $ P<0.05 vs. anti-HLAI-treated cells with halofuginone and everolimus. HLAI, human leukocyte antigen class I; ICAM-1, intracellular adhesion molecule-1; Hal, halofuginone; Ever, everolimus; Ctrl, control.

Article Snippet: Blots were incubated at 4°C for 16 h with the primary antibodies specific against activated cleaved caspase-3 (cleaved caspase-3, 1:1,000, cat. no ab13847, Abcam), focal adhesion kinase (FAK, 1:100, cat. no sc-271126, Santa Cruz Biotechnology, Inc.), phosphorylated at Tyr397 FAK (p-FAK, 1:1,000, cat. no 8556, Cell Signaling Technology, Inc.), mTOR (1:100, cat. no sc-517464, Santa Cruz Biotechnology, Inc.), phosphorylated at Ser2448 mTOR (p-mTOR, 1:100, cat. no sc-293133, Santa Cruz Biotechnology, Inc.), p70S6 kinase (p70S6K, 1:100, cat. no sc-8418, Santa Cruz Biotechnology, Inc.), phosphorylated at Thr389 p70S6K (p-p70S6K, 1:1,000, cat. no 9234, Cell Signaling Technology), protein kinase B (Akt, 1:100, cat. no sc-5298, Santa Cruz Biotechnology, Inc.), phosphorylated at Ser474 Akt (p-Akt, 1:1,000, cat. no 4060, Cell Signaling Technology, Inc.), GCN2 kinase (GCN2K, 1:100, cat. no sc-374609, Santa Cruz Biotechnology, Inc.), phosphorylated at Thr899 GCN2K (p-GCN2K, 1:1,000, cat. no ab75836; Abcam), eIF2α (1:100, cat. no sc-133132, Cell Signaling Technology, Inc.), phosphorylated at Ser51 eIF2α (p-eIF2a, 1:1,000, cat. no 9721, Cell Signaling Technology, Inc.), intercellular adhesion molecule 1 (ICAM-1, 1:1,000, cat. no 4915; Cell Signaling Technology), HLA-DR (Ultra-LEAFTM Purified anti-human HLA-DR Antibody, cat. no 307648, Biolegend), CD46 (1:1,000, cat. no CSB-PA923298, Cusabio), CD59 (1:1,000, cat. no CSB-PA004947YA01HU, Cusabio), and β-actin (1:5,000, cat no. 4967, Cell Signaling Technology, Inc.).

Techniques: Control

Journal: Journal of Virology

Article Title: Porcine Complement Regulatory Protein CD46 Is a Major Receptor for Atypical Porcine Pestivirus but Not for Classical Swine Fever Virus

doi: 10.1128/JVI.02186-20

Figure Lengend Snippet: Characterization of porcine cell lines with regard to their CD46 pig expression. (A) Phenotypical characterization of porcine cell lines by immunofluorescence staining using a CD46 pig -specific mab (green, MCA2310GA) and DAPI (blue). Asterisks (*) indicate cell lines subjected to conventional RT-PCR for subsequent sequencing. (B) Immunofluorescence staining of APPV P100 (porcine APPV-specific antiserum, red), CD46 pig (green, MCA2310GA), and DAPI (blue) at 72 h after infection of NPTr cells. (C) Strategy used for genetic characterization and manipulation of the CD46 pig gene locus. The CD46 pig -encoding mRNA was amplified by two RT-PCRs (101/710 and 604/1192) for subsequent cloning and sequencing. Absence of a CD46 pig -encoding mRNA in porcine lymphoma cell line 38A 1 D was confirmed by RT-PCRs targeting the individual CD46 pig domains (primer pairs: 192/353, 387/543, 571/710, 766/937). Positions of signal peptide (SP), complement control proteins 1 to 4 (ccp1-4), serine, threonine, proline-rich region (STP), and transmembrane domain (TM) encoded by the mRNA are depicted. In addition, positions of guide RNAs (gRNA CD46-2 and -7) used for construction of CD46 pig knockout cells are indicated (for details see ).

Article Snippet: Staining was performed using commercially available mabs against CD46 pig (MCA2310GA and MCA2262GA, Bio-Rad, 1:500 dilution) and a secondary mab Alexa fluor 488 goat anti-mouse IgG (A11029, Invitrogen, 1:1,000 dilution).

Techniques: Expressing, Immunofluorescence, Staining, Reverse Transcription Polymerase Chain Reaction, Sequencing, Infection, Amplification, Cloning, Control, Knock-Out

Journal: Journal of Virology

Article Title: Porcine Complement Regulatory Protein CD46 Is a Major Receptor for Atypical Porcine Pestivirus but Not for Classical Swine Fever Virus

doi: 10.1128/JVI.02186-20

Figure Lengend Snippet: Characterization of genetically engineered CD46 pig knockout cells. (A) Phenotypical characterization of CD46 pig knockout cells by immunofluorescence staining using a mab against CD46 pig (green, MCA2310GA) and DAPI (blue). Immunofluorescence staining of CD46 pig (green) from wild-type (WT) cell lines served as a control and is shown in . (B) CRISPR/Cas9 induced genome alterations on both alleles characterized by sequencing of plasmids containing PCR amplicons flanking target sites of the guide RNAs (primers 101fw/710rev). Consensus nucleotide sequences and deduced amino acid sequences of the regions encoding the C terminus of SP and the N terminus of ccp1 are shown. For comparison, nucleotide and deduced CD46 pig amino acid sequences of WT as determined for SPEV and PK15 cells are given in the top row. The border between SP/ccp1 and position of gRNAs including respective protospacer adjacent motifs (PAM, boxed) are indicated. For selected engineered CD46 pig knockout cell lines (ΔCD46) the corresponding sequences including deletions (Δ nt) and insertions (+ nt) are shown below the WT CD46 sequence.

Article Snippet: Staining was performed using commercially available mabs against CD46 pig (MCA2310GA and MCA2262GA, Bio-Rad, 1:500 dilution) and a secondary mab Alexa fluor 488 goat anti-mouse IgG (A11029, Invitrogen, 1:1,000 dilution).

Techniques: Knock-Out, Immunofluorescence, Staining, Control, CRISPR, Sequencing, Comparison

Journal: Journal of Virology

Article Title: Porcine Complement Regulatory Protein CD46 Is a Major Receptor for Atypical Porcine Pestivirus but Not for Classical Swine Fever Virus

doi: 10.1128/JVI.02186-20

Figure Lengend Snippet: Primers used in this study

Article Snippet: Staining was performed using commercially available mabs against CD46 pig (MCA2310GA and MCA2262GA, Bio-Rad, 1:500 dilution) and a secondary mab Alexa fluor 488 goat anti-mouse IgG (A11029, Invitrogen, 1:1,000 dilution).

Techniques: Sequencing, Plasmid Preparation

Journal: Journal of Virology

Article Title: Porcine Complement Regulatory Protein CD46 Is a Major Receptor for Atypical Porcine Pestivirus but Not for Classical Swine Fever Virus

doi: 10.1128/JVI.02186-20

Figure Lengend Snippet: Relevance of CD46 pig for the entry of porcine pestiviruses. Wild-type (WT) SPEV and PK15 as well as CD46 pig knockout cell lines (SPEVΔCD46 clones 2 and 7 and PK15ΔCD46 clone 2) were infected with APPV P17 , APPV P100 , BuPV, and CSFV strains Alfort-Tübingen (AlfT), Diepholz, Riems, Koslov, and Paderborn at an MOI of 1, respectively. Immunofluorescence staining was performed at 72 h p.i. using porcine APPV-specific antiserum, a porcine BuPV-specific antiserum, and a mab against CSFV, respectively. A strong reduction of APPV infection is evident on all SPEVΔCD46 cell lines in comparison to that on SPEV cells. PK15 cells display significantly lower permissivity to APPV P100 compared to that of SPEV cells. Non-culture-adapted APPV P17 obtained from early passage revealed the same CD46 pig dependency as the culture-adapted variant (APPV P100 ). With regard to infections with CSFV and BuPV, there are no differences in permissivity between the WT and the CD46 pig knockout cell lines.

Article Snippet: Staining was performed using commercially available mabs against CD46 pig (MCA2310GA and MCA2262GA, Bio-Rad, 1:500 dilution) and a secondary mab Alexa fluor 488 goat anti-mouse IgG (A11029, Invitrogen, 1:1,000 dilution).

Techniques: Knock-Out, Clone Assay, Infection, Immunofluorescence, Staining, Comparison, Variant Assay

Journal: Journal of Virology

Article Title: Porcine Complement Regulatory Protein CD46 Is a Major Receptor for Atypical Porcine Pestivirus but Not for Classical Swine Fever Virus

doi: 10.1128/JVI.02186-20

Figure Lengend Snippet: Production of infectious particles and RNA replication of porcine pestiviruses in dependence on CD46 pig . Wild-type (WT) SPEV and PK15, as well as CD46 pig knockout cell lines (SPEVΔCD46 clones 2 and 7 and PK15ΔCD46 clone 2), were infected with APPV P100 , CSFV Alfort-Tübingen (AlfT), and BuPV at an MOI of 1, respectively. (A) Supernatants were harvested 72 h p.i. to determine virus titers by using endpoint dilution assays in quadruplicates and in three repetitions. (B) Cells were collected at 72 h p.i. for RNA preparation and subsequent RT-PCR analysis. TaqMan based qRT-PCR assays were used for detection of CSFV and APPV genomes, whereas a SYBR green-based real-time RT-PCR was performed for detection of BuPV genomes. 30 ng total RNA was used per reaction. Samples collected from three individual experiments were tested in duplicates. Mean values with standard deviations are shown. APPV genome copy numbers obtained from WT cells are significantly higher compared to those from CD46 pig knockout cells (***, P < 0.0001, highly significant; *, P < 0.01, significant). CSFV and BuPV genome levels obtained from WT cells did not show significant differences compared to genome loads detected in infected knockout cells.

Article Snippet: Staining was performed using commercially available mabs against CD46 pig (MCA2310GA and MCA2262GA, Bio-Rad, 1:500 dilution) and a secondary mab Alexa fluor 488 goat anti-mouse IgG (A11029, Invitrogen, 1:1,000 dilution).

Techniques: Knock-Out, Clone Assay, Infection, Virus, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, SYBR Green Assay

Journal: Journal of Virology

Article Title: Porcine Complement Regulatory Protein CD46 Is a Major Receptor for Atypical Porcine Pestivirus but Not for Classical Swine Fever Virus

doi: 10.1128/JVI.02186-20

Figure Lengend Snippet: Impact of CD46 pig at early time points of porcine pestivirus infections. (A) Immunofluorescence analysis of CSFV- and BuPV-infected cells. Wild-type (WT) PK15 and PK15ΔCD46 clone 2 cells were infected with CSFV strains Alfort-Tübingen (AlfT), Diepholz, Riems, Koslov, Paderborn, and BuPV at an MOI of 1. Infections with different CSFV strains and BuPV showed no dependency on CD46 pig even very early after infection (16 h p.i.). (B) Fluorescence in situ hybridization (FISH) analysis of APPV-infected cells. WT SPEV and PK15 as well as CD46 pig knockout cell lines (SPEVΔCD46 clone 2 and PK15ΔCD46 clone 2) were infected with cell culture-adapted APPV P100 at an MOI of 0.5. Scale bars indicate 100 µm for lower magnification and 50 µm for higher magnification. A strong reduction of APPV P100 infection is evident on both CD46 pig knockout cell lines in comparison to that on WT cells at early time point of infection (16 h p.i.). APPV P100 genomes were observed only on single CD46 pig knockout cells within the infected wells. APPV P100 infection of CD46 pig -expressing WT SPEV cells at a later time point (72 h p.i.) and noninfected SPEV cells (NIC) served as controls.

Article Snippet: Staining was performed using commercially available mabs against CD46 pig (MCA2310GA and MCA2262GA, Bio-Rad, 1:500 dilution) and a secondary mab Alexa fluor 488 goat anti-mouse IgG (A11029, Invitrogen, 1:1,000 dilution).

Techniques: Immunofluorescence, Infection, Fluorescence, In Situ Hybridization, Knock-Out, Cell Culture, Comparison, Expressing

Journal: Journal of Virology

Article Title: Porcine Complement Regulatory Protein CD46 Is a Major Receptor for Atypical Porcine Pestivirus but Not for Classical Swine Fever Virus

doi: 10.1128/JVI.02186-20

Figure Lengend Snippet: Comparison of E2 envelope protein sequences of pestiviruses. (A) Phylogenetic tree (maximum likelihood) based on E2 amino acid sequences of known pestivirus species (APPV: AUL76967 ; bat: AFK85014 , AYV99177 ; rodent: ATP66856 , ATP66857 , YP009109567; pangolin: QIE06437 ; LindaV: YP009407716; whale: MK910228 ; BuPV: YP008992092; BDV: AAC16444 ; Aydin: YP006860588; ovine Italy: MG770617 ; giraffe: NP620053; pronghorn: YP009026415; BVDV-1: Q01499 ; BVDV-2: YP009513240; BVDV-3: AB871953 ; CSFV: YP009508222). APPV and CSFV sequence (bold) are the same as shown in the alignment. (B) Alignment (ClustalW) of APPV (isolate L277) and CSFV (Alfort 187) E2 amino acid sequences. Highlighted is the CSFV sequence analogous to the motif in the E2 of BVDV folding into a hairpin that might serve as ligand to the CD46 bov receptor . The positions of two nonsynonymous mutations (N751K and D752N) which occurred during cell culture adaptation of APPV are highlighted by a box.

Article Snippet: Staining was performed using commercially available mabs against CD46 pig (MCA2310GA and MCA2262GA, Bio-Rad, 1:500 dilution) and a secondary mab Alexa fluor 488 goat anti-mouse IgG (A11029, Invitrogen, 1:1,000 dilution).

Techniques: Comparison, Sequencing, Cell Culture

Journal: BMC Biology

Article Title: Genome-scale CRISPR screening at high sensitivity with an empirically designed sgRNA library

doi: 10.1186/s12915-020-00905-1

Figure Lengend Snippet: Selected Cas9-expressing single cell clones show stronger editing efficiency compared to a Cas9 bulk population. a Workflow for the selection of Cas9 single-cell clones (SCCs). SCCs were sorted from the HAP1 Cas9 bulk population and further characterized. Cas9 editing was assessed by cell surface marker knockout followed by FACS staining and cell viability upon knockout of a core essential gene. Two highly editing single-cell clones (SCC11 and SCC12) were selected for further experiments. b HAP1 Cas9 bulk, Cas9 SCC11, and Cas9 SCC12 cells were transfected with the HDCRISPRv1 vector encoding an sgRNA targeting either the safe harbor locus AAVS1 as a control or the core essential gene RNA Polymerase 2 subunit E ( POLR2E ). Editing efficiency based on cell viability of sgPOLR2E-transfected cells in comparison to sgAAVS1 control cells was addressed by crystal violet staining. The number of surviving cells was strongly reduced in cells transfected with an sgRNA directed against POLR2E ( n = 3 for each cell line and sgRNA). c Editing efficiency was furthermore assessed upon transduction of HAP1 Cas9 bulk, Cas9 SCC11, and Cas9 SCC12 cells with the HDCRISPRv1 vector expressing sgRNAs targeting the surface marker CD46 , followed by FACS staining of residual CD46 protein to address knockout efficiency. Antibody staining of the non-edited cell lines was used as a control. Lines represent the mean of independent measurements ( n = 3 for each cell line and condition)

Article Snippet: Five days after transduction, cells were harvested and the respective surface markers stained with an APC anti-human CD46 antibody (Biozol Diagnostica, Cat. No. 352405, RRID AB_2564356) or an APC anti-human CD81 antibody (Biozol Diagnostica, Cat. No BLD-349510, RRID AB_2564021).

Techniques: Expressing, Clone Assay, Selection, Marker, Knock-Out, Staining, Transfection, Plasmid Preparation, Transduction

Journal:

Article Title: Mechanism of CD150 (SLAM) Down Regulation from the Host Cell Surface by Measles Virus Hemagglutinin Protein

doi: 10.1128/JVI.78.18.9666-9674.2004

Figure Lengend Snippet: The surface expression of SLAM/CD150 is down regulated by measles virus infection. (A) Activated marmoset B-cell line B95-8 was infected with Montefiore measles virus. At 24 and 48 h p.i., SLAM surface expression was analyzed by FACS. (B) Marmoset B95-8 cells were infected with Edmonston measles virus. At 24 and 48 h p.i., SLAM surface expression was analyzed by FACS. (C) EBV-transformed human B-cell line 1A2 was infected with Montefiore measles virus. At 24 and 48 h p.i., SLAM expression was analyzed by FACS. (D) Human 1A2 cells were infected with Edmonston measles virus. At 24 and 48 h p.i., SLAM expression was determined by FACS. (E) Human 1A2 cells were infected with Montefiore measles virus. At 24 and 48 h p.i., CD46 expression was analyzed by FACS. (F) Human 1A2 cells were infected with Edmonston measles virus. At 24 and 48 h p.i., CD46 expression was analyzed by FACS. Grey lines, mock-infected cells stained with the anti-SLAM antibody (A to D) or anti-CD46 antibody (E and F) and detected with the FITC-conjugated goat anti-mouse antibody; black lines, mock-infected cells incubated with the FITC-conjugated goat anti-mouse secondary antibody only; solid peaks, cells infected with Montefiore (A, C, and E) or Edmonston (B, D, and F) measles virus stained with the anti-SLAM antibody (A to D) or the anti-CD46 antibody (E and F), followed by an FITC-conjugated goat anti-mouse antibody. Insets, levels of H protein expression on the surfaces of B95-8 and 1A2 cells infected with Montefiore 89 and Edmonston strains of measles virus following 48 h of incubation. The cells were stained with anti-measles H antibody, followed by FITC-conjugated goat anti-mouse secondary antibody. The solid lines represent infected cells; the dashed lines represent mock-infected cells.

Article Snippet: After a 5-min centrifugation at 10,000 × g , 30 μl of anti-HA affinity matrix (Roche) or 10 μl of anti-measles virus H monoclonal antibody (Chemicon) or 10 μl of an anti-CD46 monoclonal antibody (Seikagaku) was added to the supernatant, which was incubated overnight at 4°C.

Techniques: Expressing, Infection, Transformation Assay, Staining, Incubation

Journal:

Article Title: Mechanism of CD150 (SLAM) Down Regulation from the Host Cell Surface by Measles Virus Hemagglutinin Protein

doi: 10.1128/JVI.78.18.9666-9674.2004

Figure Lengend Snippet: The expression of the measles virus H protein alone can down regulate surface expression of SLAM and CD46. Human 1A2 B cells were infected with vaccinia virus recombinants that expressed Edmonston F (Ed F), Edmonston H (Ed H), or Montefiore H (Wt H) proteins. FACS analyses of SLAM surface expression (A) and CD46 surface expression (B) were performed. Black line, 1A2 cells infected with wild-type vaccinia virus (VV) incubated with an FITC-conjugated goat anti-mouse secondary antibody; gray line, 1A2 cells infected with wild-type vaccinia virus stained with a mouse anti-SLAM antibody (A) or mouse anti-CD46 antibody (B) and detected with an FITC-conjugated goat anti-mouse antibody; solid peak, 1A2 cells infected with the indicated vaccinia virus recombinants, stained with a mouse anti-SLAM antibody (A) or mouse anti-CD46 antibody (B), and detected with an FITC-conjugated goat anti-mouse secondary antibody. Infected 1A2 cells were stained for surface expression of the F and H proteins (black line) with a rabbit polyclonal antibody specific for the F protein and a monoclonal antibody directed against measles virus H proteins (C). The dashed lines represent wild-type vaccinia virus-infected cells probed for expression of the F and H proteins.

Article Snippet: After a 5-min centrifugation at 10,000 × g , 30 μl of anti-HA affinity matrix (Roche) or 10 μl of anti-measles virus H monoclonal antibody (Chemicon) or 10 μl of an anti-CD46 monoclonal antibody (Seikagaku) was added to the supernatant, which was incubated overnight at 4°C.

Techniques: Expressing, Infection, Incubation, Staining

Journal:

Article Title: Mechanism of CD150 (SLAM) Down Regulation from the Host Cell Surface by Measles Virus Hemagglutinin Protein

doi: 10.1128/JVI.78.18.9666-9674.2004

Figure Lengend Snippet: Expression of a measles virus H that is retained in the ER induces SLAM down regulation. Human 1A2 B cells were infected with normal vaccinia virus, the vaccinia-EdH recombinant virus (V-EdH), or the vaccinia-EdHER recombinant virus for 36 h. These samples were subsequently split into two samples and analyzed by FACS and immunoblot detection. (A) FACS analysis of SLAM expression on 1A2 cells infected for 36 h with vaccinia-EdH. (B) FACS analysis of SLAM expression on 1A2 cells infected for 36 h with vaccinia-EdHER. (C) FACS analysis of CD46 expression on 1A2 cells infected for 36 h with vaccinia-EdH. (D) FACS analysis of CD46 expression on 1A2 cells infected for 36 h with vaccinia-EdHER. (E) FACS analysis of H expression on 1A2 cells infected for 36 h with vaccinia-EdH. Inset, intracellular H staining of permeabilized cells with a monoclonal antibody specific for the H protein. (F) FACS analysis of H expression on 1A2 cells infected for 36 h with vaccinia-EdHER. Inset, intracellular H staining of permeabilized cells with a monoclonal antibody specific for the H protein. (G) Immunoblot analysis of measles virus H and HER expression in 1A2 cells. The 1A2 cells were lysed in SDS protein running buffer containing β-mercaptoethanol and incubated with or without Endo H for 1 h at 37°C. The samples were then subjected to SDS-PAGE and probed with a rabbit polyclonal anti-measles virus H primary antibody and detected with a peroxidase-conjugated goat anti-rabbit secondary antibody by enhanced chemiluminescence. Arrows, protein products derived from Endo H-resistant and Endo H-sensitive bands. The H protein that is retained in the ER (HER) is completely sensitive to Endo H digestion.

Article Snippet: After a 5-min centrifugation at 10,000 × g , 30 μl of anti-HA affinity matrix (Roche) or 10 μl of anti-measles virus H monoclonal antibody (Chemicon) or 10 μl of an anti-CD46 monoclonal antibody (Seikagaku) was added to the supernatant, which was incubated overnight at 4°C.

Techniques: Expressing, Infection, Recombinant, Western Blot, Staining, Incubation, SDS Page, Derivative Assay

Journal:

Article Title: Mechanism of CD150 (SLAM) Down Regulation from the Host Cell Surface by Measles Virus Hemagglutinin Protein

doi: 10.1128/JVI.78.18.9666-9674.2004

Figure Lengend Snippet: SLAM has a more rapid cell surface turnover rate than CD46. Human 1A2 B-cell lymphoma cells were treated with tunicamycin, which inhibits N-linked glycosylation and migration of newly synthesized SLAM to the cell surface. At the times indicated, FACS analyses of SLAM and CD46 expression were performed. The mean fluorescence intensity was measured by gating on live cells and was expressed as the relative percentage of steady-state receptor expression on 1A2 cells (0 h). The data shown are the averages of three independent experiments, with the error bars representing the standard deviations.

Article Snippet: After a 5-min centrifugation at 10,000 × g , 30 μl of anti-HA affinity matrix (Roche) or 10 μl of anti-measles virus H monoclonal antibody (Chemicon) or 10 μl of an anti-CD46 monoclonal antibody (Seikagaku) was added to the supernatant, which was incubated overnight at 4°C.

Techniques: Migration, Synthesized, Expressing, Fluorescence

Journal:

Article Title: Mechanism of CD150 (SLAM) Down Regulation from the Host Cell Surface by Measles Virus Hemagglutinin Protein

doi: 10.1128/JVI.78.18.9666-9674.2004

Figure Lengend Snippet: The presence of the measles virus H (MVH) protein in the ER slows or prevents the complex glycosylation of SLAM but not CD46. Human 293Tad embryonic kidney cells were transfected with pcDNA3.1-SLAM-HA3 and either pCG, pCG-H, or pCG-HER. At 36 h posttransfection, cells were lysed and immunoprecipitated with anti-H, anti-CD46, or anti-HA tag antibodies. The resulting samples were left untreated (−) or treated with Endo H or PNGaseF (+). The samples were subjected to SDS-PAGE; transferred to nitrocellulose; probed with an anti-HA antibody, anti-H antibody, or anti-CD46 antibody; and detected with a peroxidase-conjugated secondary antibody by enhanced chemiluminescence. (A) Lysates were immunoprecipitated (IP) with an anti-HA antibody, and the blots were probed with an anti-HA tag antibody. Expression of H or HER inhibits complex glycosylation of SLAM and maintains its sensitivity to Endo H. (B) Lysates were immunoprecipitated with a mouse monoclonal anti-CD46 antibody that recognized endogenous CD46. The blots were probed with a rabbit polyclonal anti-CD46 antibody. Expression of H or HER did not inhibit the complex glycosylation of CD46, and the glycoprotein exhibited little or no sensitivity to Endo H. (C) Lysates were immunoprecipitated with a mouse monoclonal anti-MVH antibody, and the blots were probed with an anti-HA antibody. The SLAM receptor coprecipitates with MVH proteins, and HER expression prevents the complex glycosylation of SLAM and maintains its sensitivity to Endo H. (D) Lysates were immunoprecipitated with an anti-HA antibody, and the blots were probed with a rabbit polyclonal anti-MVH antibody. MVH coprecipitates with SLAM and is partially sensitive to Endo H. (E) 293Tad cells were transfected with only pCG, pCG-H, or pCG-HER. Lysates were immunoprecipitated with a mouse monoclonal anti-MVH antibody, and the blots were probed with a rabbit polyclonal anti-CD46 antibody as in panel B. Endogenous CD46 does not coprecipitate with MVH. (F) 293Tad cells were transfected with only pCG, pCG-H, or pCG-HER. Lysates were immunoprecipitated with a mouse monoclonal anti-CD46 antibody, and the blots were probed with a rabbit polyclonal anti-MVH antibody as in panel D. MVH does not coprecipitate with endogenous CD46.

Article Snippet: After a 5-min centrifugation at 10,000 × g , 30 μl of anti-HA affinity matrix (Roche) or 10 μl of anti-measles virus H monoclonal antibody (Chemicon) or 10 μl of an anti-CD46 monoclonal antibody (Seikagaku) was added to the supernatant, which was incubated overnight at 4°C.

Techniques: Transfection, Immunoprecipitation, SDS Page, Expressing

Journal:

Article Title: Mechanism of CD150 (SLAM) Down Regulation from the Host Cell Surface by Measles Virus Hemagglutinin Protein

doi: 10.1128/JVI.78.18.9666-9674.2004

Figure Lengend Snippet: Down regulation of SLAM and CD46 from the surfaces of 1A2 cells results from coincubation with Sf9 insect and Chinese hamster ovary (CHOP) cells that express measles virus H on their surfaces. (A) CHOP cells were transfected with pcDNA1.1-H. At 24 h after transfection, the CHOP cells were washed once with PBS, and 5 × 105 1A2 cells were added. FACS analysis of SLAM and CD46 expression on 1A2 cells was performed at 0 (gray line) and 24 h (solid peak) after coincubation. FACS analysis of H expression on the CHOP cells was also performed in the right graph using a monoclonal antibody that recognizes measles virus H. Gray line, cells transfected with pcDNA1.1; solid peak, cells transfected with pcDNA1.1-H. 1A2 cells were stained and gated for CD21, a marker specific for B cells. CHOP cells that express Edmonston H on their surfaces down regulate expression of CD46 and SLAM from the surfaces of human 1A2 cells. (B) Sf9 insect cells were infected with wild-type baculovirus or a recombinant baculovirus that expresses the Edmonston H protein. At 18 h after infection, the Sf9 cells were added to 1A2 cells at the ratios indicated. FACS analysis of SLAM expression on the 1A2 cells and H expression on the Sf9 cells was performed 24 h after coincubation. In the left column, the black line represents 1A2 cells stained with a goat anti-mouse secondary antibody, the gray line represents 1A2 cells coincubated with Sf9 cells infected with wild-type baculovirus and stained with mouse anti-human SLAM, and the solid peak represents 1A2 cells coincubated with Sf9 cells expressing H and stained with mouse anti-human SLAM. In the right column, the solid peak represents Sf9 insect cells expressing H protein stained with mouse anti-measles virus H and the gray line represents Sf9 insect cells infected with wild-type baculovirus stained with mouse anti-measles virus H.

Article Snippet: After a 5-min centrifugation at 10,000 × g , 30 μl of anti-HA affinity matrix (Roche) or 10 μl of anti-measles virus H monoclonal antibody (Chemicon) or 10 μl of an anti-CD46 monoclonal antibody (Seikagaku) was added to the supernatant, which was incubated overnight at 4°C.

Techniques: Transfection, Expressing, Staining, Marker, Infection, Recombinant

Journal: Immunology

Article Title: Co-ordinated regulation of plasmacytoid dendritic cell surface receptors upon stimulation with herpes simplex virus type 1

doi: 10.1111/j.1365-2567.2009.03176.x

Figure Lengend Snippet: Flow cytometry analysis of the expression and regulation of plasmacytoid dendritic cell (PDC) surface receptors. Representative dot plot and histogram analysis of the expression of CD319 in PDC cultivated in medium containing interleukin-3 (IL-3; black) and additionally exposed to UV-inactivated herpes simplex virus type 1 (HSVUV; red) for 40 hr. Surface receptor expression determined in uncultivated PDC directly after the isolation of peripheral blood mononuclear cells (PBMC) (open diamonds) or after cultivation in IL-3 (black diamonds), IL-3/HSVUV (red diamonds), or HSVUV (blue diamonds). The mean fluorescence intensities (MFI) of a total of 35 donors are presented on a logarithmic scale (log10). After correction for multiple comparisons (Bonferroni; n = 51), P values ≤ 0·05 are indicated as horizontal bars. The grey lines represent isotype control values. Only those receptors are presented which were significantly regulated upon IL-3 and/or HSV-1 exposure. A total of 18 receptors were found to be expressed but not regulated (CD2, CD8, CD18, CD44, CD46, CD48, CD50, CD58, CD59, CD66a, CD80, CD82, CD83, CD97, CD162, CD170, HLA-DR, HLA-ABC).

Article Snippet: Antibodies against the following surface antigens were purchased from Immunotools (Friesoythe, Germany): CD2, CD9, CD11a, CD14, CD18, CD29, CD31, CD33, CD36, CD37, CD38, CD43, CD44, CD46, CD48, CD49d, CD50, CD54, CD55, CD58, CD59, CD62L, CD63, CD66acde, CD74, CD80, CD95, CD97, CD99R, CD103, HLA-DR, all FITC-conjugated; CD69-PE, and CD14-PE/Cy5; CD26, CD53 and CD162 were unconjugated.

Techniques: Flow Cytometry, Expressing, Virus, Isolation, Fluorescence, Control