Journal: eLife

Article Title: Shore crabs reveal novel evolutionary attributes of the mushroom body

doi: 10.7554/eLife.65167

Figure Lengend Snippet: ( A, B ). Amira-generated 3D reconstructions of the gyri and their calyces. Black arrows indicate the direction of the eyestalk nerve; likewise in panels C–H . ( C ) Ensemble of efferent dendrites in gyrus 2 associated with calyx 1 (Ca1Gy2). The represented area is boxed in panel B . ( D ) Afferent tributary from the olfactory globular tract (olfactory projection neurons) supplying gyrus Ca1Gy1 neuropil (open rectangle in panel A ). ( E ) Smooth oval presynaptic specializations denote the terminals of another olfactory globular tract tributary that exclusively supplies the most proximal gyrus (pGy, rectangle in panel A ), which has no obvious connection to the mushroom body. ( F ) Double-labeling with anti–GAD (yellow) and anti-allatostatin (cyan) of the lateral protocerebrum resolves allatostatin immunoreactivity in the proximal gyrus (rectangle in A and F ), which shows little to no GAD immunoreactivity. ( G, H ) Stacked z-axis projections of Golgi-impregnated tracts, imaged through two successive 50 μm sections showing massive supply by afferent neurons. These reach levels of the lateral protocerebrum that include those at and well beneath the gyriform layer (box I, panel G ), including levels of the calyces (box J in panel G and box K in panel H ). Notably, many efferent neurons relaying information from gyri (panel H ) send their downstream axons from the lateral protocerebrum into these tracts. ( I–K ) Enlargements from regions indicated in G , H , but at sample thicknesses of less than 20 μm to isolate details obscured in flattened optical sections. Scale bars, A–C , F–H , 100 μm; D , E , 50 μm; I–K , 50 μm.

Article Snippet: Antibody , Allatostatin (Ast7; Mouse, monoclonal) , Developmental Studies Hybridoma Bank (DSHB) , CAT#: 5F10; RRID: AB_528076 , 1:100.

Techniques: Generated, Labeling

Journal: eLife

Article Title: Shore crabs reveal novel evolutionary attributes of the mushroom body

doi: 10.7554/eLife.65167

Figure Lengend Snippet:

Article Snippet: Antibody , Allatostatin (Ast7; Mouse, monoclonal) , Developmental Studies Hybridoma Bank (DSHB) , CAT#: 5F10; RRID: AB_528076 , 1:100.

Techniques: Staining, Purification, Electron Microscopy, Software, Microscopy, Light Microscopy

Journal: PLOS One

Article Title: Kummerowia striata extract protects paracetamol-induced liver injury by modulating the S1P/Nrf2/Keap1 pathway

doi: 10.1371/journal.pone.0329837

Figure Lengend Snippet: (A-C) Immunohistochemical (IHC) staining of liver tissue for NRF2, Keap1, and SPHK1. (D, E) Western blot analysis of protein levels for NQO1 and HO-1 following NRF2 nuclear translocation.(F, G) mRNA level changes of downstream targets NQO1 and HO-1 following NRF2 nuclear translocation.(H, J, L) Changes in mRNA levels and protein content of SPHK1, along with Western blot analysis of protein expression.(I) Changes in S1P levels.(K) Changes in SPHK1 protein levels.(M-Q) mRNA expression levels of S1PR1–5.(R, S) Changes in protein levels of S1PR2 and S1PR4.* P < 0.05, ** P < 0.01, *** P < 0.001 indicate statistically significant differences compared with the control or model group.

Article Snippet: 4% Tissue Cell Fixative (4% Paraformaldehyde) (purchased from: Solarbio; Catalog No.: P1110); Alanine Aminotransferase (ALT) Assay Kit (IFCC method) (purchased from: Mindray; Catalog No.: 140124009); Aspartate Aminotransferase (AST) Assay Kit (IFCC method) (purchased from: Mindray; Catalog No.: 140224004); Lactate Dehydrogenase (LDH) Assay Kit (IFCC method) (purchased from: Mindray; Catalog No.: 142723011);Total Superoxide Dismutase (T-SOD) Assay Kit (WST-1 method) (purchased from: Nanjing Jiancheng; Catalog No.: A001-3–2);Malondialdehyde (MDA) Assay Kit;Reduced Glutathione (GSH) Assay Kit (Microplate method) (purchased from: Nanjing Jiancheng; Catalog No.: A006-2–1);Mouse Interleukin-6 (IL-6) ELISA Quantitative Detection Kit (purchased from: Proteintech; Catalog No.: KE10007); Mouse Interleukin-1 Beta (IL-1β) ELISA Kit (purchased from: Elabscience; Catalog No.: E-EL-M0037); Mouse Tumor Necrosis Factor Alpha (TNF-α) ELISA Kit (purchased from: Elabscience; Catalog No.: E-EL-M3063); Mouse Interleukin-10 (IL-10) ELISA Kit (purchased from: BY Abscience; Catalog No.: BY- EM220162 ); Sphingosine 1-Phosphate (S1P) Assay Kit (purchased from: Echelon Biosciences; Catalog No.: K-1900); Mouse Sphingosine Kinase-2 (Sphk-2) ELISA Quantitative Detection Kit (purchased from: UpingBio; Catalog No.: SYP-M2060); Mouse SPHK1 ELISA Kit (purchased from: UpingBio; Catalog No.: SYP-M2057); Mouse S1PR2 ELISA Kit (purchased from: UpingBio; Catalog No.: SYP-M1458); Mouse S1PR4 ELISA Kit (purchased from: BY Abscience; Catalog No.: BYHS505577); Mouse Catalase (CAT) ELISA Kit (purchased from: Jinhengnuo; Catalog No.: 231024JN); Mouse Keap1 ELISA Kit (purchased from: WeiaoBio; Catalog No.: EM30387XS); SPHK1 Polyclonal Antibody (purchased from: Proteintech; Catalog No.: 10670–1-AP); NFE2L2 (Nrf2) Recombinant Antibody (purchased from: Proteintech; Catalog No.: 8059–1-RR); Heme Oxygenase 1 Antibody (purchased from: Abways; Catalog No.: CY8761); NQO1 Antibody (purchased from: Abways; Catalog No.: CY6710); Omni-EasyTM Ready-to-use BCA Protein Quantification Kit (purchased from: Yamei; Catalog No.: ZJ102); General Antibody Dilution Buffer (purchased from: Yamei; Catalog No.: PS119); ToloScript All-in-one RT EasyMix for qPCR (One-step Reverse Transcription) (purchased from: Tolobio; Catalog No.: 22107); 2xQ5 SYBR qPCR Master Mix (Universal) (purchased from: Tolobio; Catalog No.: 22206−01).

Techniques: Immunohistochemical staining, Immunohistochemistry, Western Blot, Translocation Assay, Expressing, Control

Journal: PLOS One

Article Title: Kummerowia striata extract protects paracetamol-induced liver injury by modulating the S1P/Nrf2/Keap1 pathway

doi: 10.1371/journal.pone.0329837

Figure Lengend Snippet: (A) A heatmap of the binding affinity of the 13 components of Ks with S1P, SPHK1, S1PR2, S1PR4, and NRF2-KEAP1, showing the best docking results. (B) Docking results of S1P with stigmasterol.(C) Docking results of SPHK1 with apigenin 7-O-glucuronide.(D) Docking results of NRF2-KEAP1 with genistin. (E) Docking results of S1PR2 with apigenin 7-O-glucuronide.(F) Docking results of S1PR4 with apigenin 7-O-glucuronide. (Specific analysis can be found in the original text.).

Article Snippet: 4% Tissue Cell Fixative (4% Paraformaldehyde) (purchased from: Solarbio; Catalog No.: P1110); Alanine Aminotransferase (ALT) Assay Kit (IFCC method) (purchased from: Mindray; Catalog No.: 140124009); Aspartate Aminotransferase (AST) Assay Kit (IFCC method) (purchased from: Mindray; Catalog No.: 140224004); Lactate Dehydrogenase (LDH) Assay Kit (IFCC method) (purchased from: Mindray; Catalog No.: 142723011);Total Superoxide Dismutase (T-SOD) Assay Kit (WST-1 method) (purchased from: Nanjing Jiancheng; Catalog No.: A001-3–2);Malondialdehyde (MDA) Assay Kit;Reduced Glutathione (GSH) Assay Kit (Microplate method) (purchased from: Nanjing Jiancheng; Catalog No.: A006-2–1);Mouse Interleukin-6 (IL-6) ELISA Quantitative Detection Kit (purchased from: Proteintech; Catalog No.: KE10007); Mouse Interleukin-1 Beta (IL-1β) ELISA Kit (purchased from: Elabscience; Catalog No.: E-EL-M0037); Mouse Tumor Necrosis Factor Alpha (TNF-α) ELISA Kit (purchased from: Elabscience; Catalog No.: E-EL-M3063); Mouse Interleukin-10 (IL-10) ELISA Kit (purchased from: BY Abscience; Catalog No.: BY- EM220162 ); Sphingosine 1-Phosphate (S1P) Assay Kit (purchased from: Echelon Biosciences; Catalog No.: K-1900); Mouse Sphingosine Kinase-2 (Sphk-2) ELISA Quantitative Detection Kit (purchased from: UpingBio; Catalog No.: SYP-M2060); Mouse SPHK1 ELISA Kit (purchased from: UpingBio; Catalog No.: SYP-M2057); Mouse S1PR2 ELISA Kit (purchased from: UpingBio; Catalog No.: SYP-M1458); Mouse S1PR4 ELISA Kit (purchased from: BY Abscience; Catalog No.: BYHS505577); Mouse Catalase (CAT) ELISA Kit (purchased from: Jinhengnuo; Catalog No.: 231024JN); Mouse Keap1 ELISA Kit (purchased from: WeiaoBio; Catalog No.: EM30387XS); SPHK1 Polyclonal Antibody (purchased from: Proteintech; Catalog No.: 10670–1-AP); NFE2L2 (Nrf2) Recombinant Antibody (purchased from: Proteintech; Catalog No.: 8059–1-RR); Heme Oxygenase 1 Antibody (purchased from: Abways; Catalog No.: CY8761); NQO1 Antibody (purchased from: Abways; Catalog No.: CY6710); Omni-EasyTM Ready-to-use BCA Protein Quantification Kit (purchased from: Yamei; Catalog No.: ZJ102); General Antibody Dilution Buffer (purchased from: Yamei; Catalog No.: PS119); ToloScript All-in-one RT EasyMix for qPCR (One-step Reverse Transcription) (purchased from: Tolobio; Catalog No.: 22107); 2xQ5 SYBR qPCR Master Mix (Universal) (purchased from: Tolobio; Catalog No.: 22206−01).

Techniques: Binding Assay

Journal: Scientific reports

Article Title: Generation of fully functional hepatocyte-like organoids from human induced pluripotent stem cells mixed with Endothelial Cells.

doi: 10.1038/s41598-019-45514-3

Figure Lengend Snippet: Figure 3. Tissue-specific gene expression analysis by Real-Time PCR. The relative quantities of tissue-specific genes were measured at the mRNA level to assess the final maturation of the terminally differentiated HLCs for both experimental conditions. The results showed an overall trend of higher expression of all markers for the hiPSC-EB + EC-HLCs when compared to the hiPSC-EB-HLCs, with statistically significant higher expression for the albumin, HNF-4α, HNF-4β, CYP1A2, CYP2B6 and UGT1A3. Almost for all the genes tested, both hiPSC-EB-HLCs and hiPSC-EB + EC-HLCs showed a gene expression that was statistically higher than the one observed in human primary hepatocyte (HPH) and human neonatal hepatocyte (HNH). HPH was used as a positive control for mature phenotype, while HNH was used as a control for immature genotype. Results for the differentiated hiPSCs for both conditions were normalized to undifferentiated hiPSCs (experimental negative control). Data presented as mean ± SD (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: We used the following human specific primary antibodies: rabbit anti SOX17 (Santa Cruz, sc-20099; 1:100); mouse anti FOXA2 (Abcam, ab60721); 5 μg ml−1, goat anti Hhex (Santa Cruz, sc-15128; 1:100); mouse anti GATA-4 (Santa Cruz, sc-25310; 1:100); mouse anti AFP (Santa Cruz, sc-166325; 1:100); mouse anti HNF-4α (Santa Cruz, sc-8987; 1:100); goat anti Albumin (Santa Cruz, Santa Cruz, CA, sc-46293; 1:100); mouse anti Cytokeratin 18 (CK-18) (Abcam, ab82254, 5 μg ml−1); mouse anti HNF1-α (Santa Cruz, sc-135939; 1:100); rabbit anti human C-MET (Santa Cruz, sc-10; 1:100), mouse anti human AGXT2L2 (ALT) (Santa Cruz, sc-365670; 1:100), mouse anti human AATM (E-7) (AST) (Santa Cruz, sc-271702; 1:100), mouse anti human AAT (H-7) (A1AT) (Santa Cruz, sc-166018; 1:100), mouse anti HNF-3β (RY-7) (Santa Cruz, sc-101060; 1:100), and Alexa Fluor 488 mouse anti-CD31 antibody (Abcam; ab215911).

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Expressing, Positive Control, Control, Negative Control

Journal: Scientific reports

Article Title: Generation of fully functional hepatocyte-like organoids from human induced pluripotent stem cells mixed with Endothelial Cells.

doi: 10.1038/s41598-019-45514-3

Figure Lengend Snippet: Figure 4. Tissue-specific marker analysis through immunofluorescence and FACS analysis. Following the differentiation program, terminally differentiated hiPSC-EB + EC-HLCs expressed mature hepatocyte-specific markers, as evidenced by the presence co-staining of (a) ALBUMIN and Alpha-1 Anti-Trypsin (A1AT), (b) ALBUMIN and ALT, (c) ALBUMIN and AST (d) ALBUMIN and CD31, (e) ALBUMIN and HNF-3β. Scale bar 100 µm. (f) FACS analysis for albumin positive cells showed a greater percentage of albumin positive cells in the condition with endothelial compared with the one without (86% vs 59%). (g) Magnified (60X) detail of ALBUMIN/CD31 positive cells showing the rosettes organization of CD31 positive cells interspersed with ALBUMIN positive cells. Scale bar 50 µm.

Article Snippet: We used the following human specific primary antibodies: rabbit anti SOX17 (Santa Cruz, sc-20099; 1:100); mouse anti FOXA2 (Abcam, ab60721); 5 μg ml−1, goat anti Hhex (Santa Cruz, sc-15128; 1:100); mouse anti GATA-4 (Santa Cruz, sc-25310; 1:100); mouse anti AFP (Santa Cruz, sc-166325; 1:100); mouse anti HNF-4α (Santa Cruz, sc-8987; 1:100); goat anti Albumin (Santa Cruz, Santa Cruz, CA, sc-46293; 1:100); mouse anti Cytokeratin 18 (CK-18) (Abcam, ab82254, 5 μg ml−1); mouse anti HNF1-α (Santa Cruz, sc-135939; 1:100); rabbit anti human C-MET (Santa Cruz, sc-10; 1:100), mouse anti human AGXT2L2 (ALT) (Santa Cruz, sc-365670; 1:100), mouse anti human AATM (E-7) (AST) (Santa Cruz, sc-271702; 1:100), mouse anti human AAT (H-7) (A1AT) (Santa Cruz, sc-166018; 1:100), mouse anti HNF-3β (RY-7) (Santa Cruz, sc-101060; 1:100), and Alexa Fluor 488 mouse anti-CD31 antibody (Abcam; ab215911).

Techniques: Marker, Immunofluorescence, Staining

Journal: Journal of Virology

Article Title: A Kaposi's Sarcoma-Associated Herpesvirus Infection Mechanism Is Independent of Integrins α3β1, αVβ3, and αVβ5

doi: 10.1128/JVI.00803-18

Figure Lengend Snippet: CRISPR-Cas9 guide RNA sequences used to target the indicated genes

Article Snippet: Heparan sulfate antibody (F58-10E4) was purchased from Amsbio; integrin α3 antibody (P1B5) was purchased from Calbiochem; integrin αV, integrin β7, EphA2, and EphA5 antibodies (MAB12191, MAB4669, AF3035, and MAB541, respectively) were obtained from R&D Systems; integrin β1 and integrin β3 antibodies (T2S/16 and PM6/13, respectively) were obtained from Novus Biologicals; integrin β5 and EphA2 antibodies (AST-3T and SHM16, respectively) were obtained from BioLegend; xCT and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies (ab37185 and ab181602, respectively) were obtained from Abcam; DC-SIGN antibody (DCN47.5) was obtained from Miltenyi Biotec; EphA4 antibody (4C8H5) was obtained from ThermoFisher; EphB2 antibody (2D12C6) was obtained from Santa Cruz Biotech; and Flag antibody (M2) was obtained from Sigma-Aldrich.

Techniques: CRISPR, Sequencing

Journal: Journal of Virology

Article Title: A Kaposi's Sarcoma-Associated Herpesvirus Infection Mechanism Is Independent of Integrins α3β1, αVβ3, and αVβ5

doi: 10.1128/JVI.00803-18

Figure Lengend Snippet: EphA4 and EphB2 are dispensable for infection of Caki-1 cells. (A, E, F, and H) A total of 120 μg (A, E, and H) or 50 μg (F) of the indicated whole-cell lysate proteins was run on a 10% SDS-PAGE gel and blotted for EphA4 (A and E) or EphB2 (F and H) and GAPDH as a loading control. (B and C) WT and EPHA4 KO Caki-1 cells (B) or WT, EPHA2 KO, and EPHA4/EPHA2 DKO Caki-1 cells (C) were infected with KSHV in triplicate, and the infection percentage was measured by flow cytometry at 2 days postinfection. The infection percentages of KO cell lines were normalized to the average infection percentage of WT cells, and data from a representative experiment are shown. (D) EPHA4 KO and EPHA4/EPHA2 DKO Caki-1 cells were immunostained for surface EphA2 expression. Gray histograms represent isotype controls. (G) WT and EPHB2 KO Caki-1 cells were infected with KSHV in triplicate, and infection percentages were quantified by flow cytometry at 2 days postinfection. The rates of infection of the KO line were normalized to the average WT infection rate, and data from a representative experiment are shown. *, P < 0.05.

Article Snippet: Heparan sulfate antibody (F58-10E4) was purchased from Amsbio; integrin α3 antibody (P1B5) was purchased from Calbiochem; integrin αV, integrin β7, EphA2, and EphA5 antibodies (MAB12191, MAB4669, AF3035, and MAB541, respectively) were obtained from R&D Systems; integrin β1 and integrin β3 antibodies (T2S/16 and PM6/13, respectively) were obtained from Novus Biologicals; integrin β5 and EphA2 antibodies (AST-3T and SHM16, respectively) were obtained from BioLegend; xCT and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies (ab37185 and ab181602, respectively) were obtained from Abcam; DC-SIGN antibody (DCN47.5) was obtained from Miltenyi Biotec; EphA4 antibody (4C8H5) was obtained from ThermoFisher; EphB2 antibody (2D12C6) was obtained from Santa Cruz Biotech; and Flag antibody (M2) was obtained from Sigma-Aldrich.

Techniques: Infection, SDS Page, Control, Flow Cytometry, Expressing

Journal: Journal of Virology

Article Title: A Kaposi's Sarcoma-Associated Herpesvirus Infection Mechanism Is Independent of Integrins α3β1, αVβ3, and αVβ5

doi: 10.1128/JVI.00803-18

Figure Lengend Snippet: Perturbations in KSHV receptor expression do not unexpectedly affect other known receptors. WT, EPHA2 KO, ITGB1 KO, and ITGAV/ITGA3 DKO Caki-1 cells were concurrently immunostained for surface expression of nontargeted, known KSHV receptors and analyzed by flow cytometry. Gray histograms represent isotype controls.

Article Snippet: Heparan sulfate antibody (F58-10E4) was purchased from Amsbio; integrin α3 antibody (P1B5) was purchased from Calbiochem; integrin αV, integrin β7, EphA2, and EphA5 antibodies (MAB12191, MAB4669, AF3035, and MAB541, respectively) were obtained from R&D Systems; integrin β1 and integrin β3 antibodies (T2S/16 and PM6/13, respectively) were obtained from Novus Biologicals; integrin β5 and EphA2 antibodies (AST-3T and SHM16, respectively) were obtained from BioLegend; xCT and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies (ab37185 and ab181602, respectively) were obtained from Abcam; DC-SIGN antibody (DCN47.5) was obtained from Miltenyi Biotec; EphA4 antibody (4C8H5) was obtained from ThermoFisher; EphB2 antibody (2D12C6) was obtained from Santa Cruz Biotech; and Flag antibody (M2) was obtained from Sigma-Aldrich.

Techniques: Expressing, Flow Cytometry

Journal: Journal of Virology

Article Title: A Kaposi's Sarcoma-Associated Herpesvirus Infection Mechanism Is Independent of Integrins α3β1, αVβ3, and αVβ5

doi: 10.1128/JVI.00803-18

Figure Lengend Snippet: EphA2 is required for infection of Caki-1 and HeLa cells. (A) WT and EPHA2 KO Caki-1 cells were immunostained for surface EphA2 expression with the SHM16 antibody and analyzed by flow cytometry. The gray histogram represents the isotype control. (B) WT and EPHA2 KO Caki-1 cells were infected with KSHV in duplicate, and infection rates were quantified by flow cytometry. The rate of infection of the EPHA2 KO pool was normalized to the average WT infection rate, and data were pooled from multiple experiments. (C) WT Caki-1 and EPHA2 KO clone A1 cells were immunostained for surface EphA2 with the antibody AF3035 and analyzed by flow cytometry. The gray histogram represents the isotype control. (D) Fifteen micrograms of whole-cell lysate protein from WT Caki-1 cells and EPHA2 KO clone A1 was run on an SDS-PAGE gel and blotted for EphA2 with AF3035 and GAPDH. (E) EPHA2 KO clone A1 Caki-1 cells were preblocked with EGFR-Fc or ephrin-A4–Fc at 10 μg/ml at 4°C and then infected in triplicate in the presence of EGFR-Fc or ephrin-A4–Fc at 5 μg/ml at 37°C. The infection percentage was measured by flow cytometry at 2 days postinfection, and percent infection was normalized to the average EPHA2 KO infection rate. (D) Mixed EPHA2 KO HeLa cells were immunostained for surface EphA2 expression with the SHM16 antibody and analyzed by flow cytometry. The gray histogram represents the isotype control. (E) Mixed EPHA2 KO HeLa cells were infected with KSHV in triplicate, and the infection percentage was measured by flow cytometry at 2 days postinfection. The cells were also immunostained for surface EphA2 and gated on EphA2-high or -low cells, as indicated in panel D. The rates of infection of EphA2-low cells were normalized to those of EphA2-high cells in each well, and data from a representative experiment are shown. *, P < 0.05.

Article Snippet: Heparan sulfate antibody (F58-10E4) was purchased from Amsbio; integrin α3 antibody (P1B5) was purchased from Calbiochem; integrin αV, integrin β7, EphA2, and EphA5 antibodies (MAB12191, MAB4669, AF3035, and MAB541, respectively) were obtained from R&D Systems; integrin β1 and integrin β3 antibodies (T2S/16 and PM6/13, respectively) were obtained from Novus Biologicals; integrin β5 and EphA2 antibodies (AST-3T and SHM16, respectively) were obtained from BioLegend; xCT and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies (ab37185 and ab181602, respectively) were obtained from Abcam; DC-SIGN antibody (DCN47.5) was obtained from Miltenyi Biotec; EphA4 antibody (4C8H5) was obtained from ThermoFisher; EphB2 antibody (2D12C6) was obtained from Santa Cruz Biotech; and Flag antibody (M2) was obtained from Sigma-Aldrich.

Techniques: Infection, Expressing, Flow Cytometry, Control, SDS Page

Journal: Journal of Virology

Article Title: A Kaposi's Sarcoma-Associated Herpesvirus Infection Mechanism Is Independent of Integrins α3β1, αVβ3, and αVβ5

doi: 10.1128/JVI.00803-18

Figure Lengend Snippet: EphA2, EphA4, and EphA5 rescue KSHV infection in EPHA2 KO Caki-1 cells. (A) Diagram of generalized full-length and PPT-3×Flag-mature ephrin receptor constructs. SAM, sterile alpha motif. (B and C) Live (B) or fixed and permeabilized (C) 3×Flag-tagged ephrin receptor-transduced EPHA2 KO cells and a vector control were immunostained for surface (B) or intracellular (C) 3×Flag expression and analyzed by flow cytometry. Gray histograms represent isotype controls. (D) The indicated cell lysates were run on 10% SDS-PAGE gels and blotted for 3×Flag, EphA4, and EphA5 with matched GAPDH as a loading control. For the Flag and EphA5 blots, 15 μg of whole-cell lysate protein was loaded. For the EphA4 blot, 120 μg of whole-cell lysate protein was loaded. (E) The indicated cell lines were immunostained for surface EphA2 or EphA5 expression and analyzed by flow cytometry. Gray histograms represent isotype controls. (F) The indicated cell lines were infected with KSHV in triplicate, and the infection rate was quantified by flow cytometry at 2 days postinfection. Data from a representative experiment are shown. (G) The 3×Flag expression histograms of infected 3×Flag-tagged ephrin receptor-transduced cell lines were divided into five successive gates, as shown. The infection rate within each gate was plotted against the fold MFI over the isotype of each gate. *, P < 0.05.

Article Snippet: Heparan sulfate antibody (F58-10E4) was purchased from Amsbio; integrin α3 antibody (P1B5) was purchased from Calbiochem; integrin αV, integrin β7, EphA2, and EphA5 antibodies (MAB12191, MAB4669, AF3035, and MAB541, respectively) were obtained from R&D Systems; integrin β1 and integrin β3 antibodies (T2S/16 and PM6/13, respectively) were obtained from Novus Biologicals; integrin β5 and EphA2 antibodies (AST-3T and SHM16, respectively) were obtained from BioLegend; xCT and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies (ab37185 and ab181602, respectively) were obtained from Abcam; DC-SIGN antibody (DCN47.5) was obtained from Miltenyi Biotec; EphA4 antibody (4C8H5) was obtained from ThermoFisher; EphB2 antibody (2D12C6) was obtained from Santa Cruz Biotech; and Flag antibody (M2) was obtained from Sigma-Aldrich.

Techniques: Infection, Construct, Sterility, Plasmid Preparation, Control, Expressing, Flow Cytometry, SDS Page

Journal: Journal of Virology

Article Title: A Kaposi's Sarcoma-Associated Herpesvirus Infection Mechanism Is Independent of Integrins α3β1, αVβ3, and αVβ5

doi: 10.1128/JVI.00803-18

Figure Lengend Snippet: The EphA2 ectodomain is sufficient to rescue the rate of infection of EPHA2 KO cells. (A) Diagram of EphA2 truncation and domain swap constructs. (B and C) WT, EPHA2 KO, and EPHA2 KO cells transduced with the EphA2 constructs indicated in panel A were infected with KSHV in triplicate, and the infection rate was quantified by flow cytometry at 2 days postinfection. The infection rates were normalized to the average rate of infection of WT cells, and data from a representative experiment are shown. (D) WT, EPHA2 KO, and the indicated transduced EPHA2 KO Caki-1 cells were immunostained for surface EphA2 expression and analyzed by flow cytometry. Gray histograms represent the isotype controls. *, P < 0.05.

Article Snippet: Heparan sulfate antibody (F58-10E4) was purchased from Amsbio; integrin α3 antibody (P1B5) was purchased from Calbiochem; integrin αV, integrin β7, EphA2, and EphA5 antibodies (MAB12191, MAB4669, AF3035, and MAB541, respectively) were obtained from R&D Systems; integrin β1 and integrin β3 antibodies (T2S/16 and PM6/13, respectively) were obtained from Novus Biologicals; integrin β5 and EphA2 antibodies (AST-3T and SHM16, respectively) were obtained from BioLegend; xCT and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies (ab37185 and ab181602, respectively) were obtained from Abcam; DC-SIGN antibody (DCN47.5) was obtained from Miltenyi Biotec; EphA4 antibody (4C8H5) was obtained from ThermoFisher; EphB2 antibody (2D12C6) was obtained from Santa Cruz Biotech; and Flag antibody (M2) was obtained from Sigma-Aldrich.

Techniques: Infection, Construct, Transduction, Flow Cytometry, Expressing

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: FPR2/ALX receptor expression and internalization are critical for lipoxin A 4 and annexin-derived peptide-stimulated phagocytosis

doi: 10.1096/fj.10-159913

Figure Lengend Snippet: LXA4- and Ac2–26-induced internalization of FPR2/ALX: confocal microscopy. Hela cells transiently expressing HA-tagged FPR2/ALX plated on poly-l-lysine-coated coverslips were treated with vehicle (EtOH), LXA4 (1 nM), or Ac2–26 (30 μM) for 15 min. Cells were fixed with paraformaldehyde, permeabilized with Triton X-100, and subjected to detection of receptor using an anti-HA primary antibody, followed by a Oregon Green-conjugated secondary antibody. Confocal microscopy images representative of ≥5 experiments are shown. Image of untransfected cells is shown as negative control.

Article Snippet: Following incubation (40 min at 4°C) with a mouse monoclonal anti-human FPR2 antibody (clone: 6C7-3; kind gift of Dr. D Anderson, Astra Zeneca, Macclesfield, UK) or an isotype control (Serotec, Oxford, UK), cells were washed twice with PBC and incubated for another 30 min at 4°C with an anti-mouse FITC-conjugated secondary antibody (clone: Star 9B; Serotec).

Techniques: Confocal Microscopy, Expressing, Negative Control

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: FPR2/ALX receptor expression and internalization are critical for lipoxin A 4 and annexin-derived peptide-stimulated phagocytosis

doi: 10.1096/fj.10-159913

Figure Lengend Snippet: Constitutive and agonist-dependent internalization of FPR2/ALX and FPR3. Hela cells transiently transfected with HA-tagged FPR2/ALX (A–C) or FPR3 (D–F) were labeled at 4°C for 1 h with anti HA primary antibody prior to incubate the cells at 37°C in the presence of vehicle (EtOh) or LXA4 (1 nM) for 30 min. Cells were then fixed with paraformaldehyde and receptor was stained with Oregon Green anti-mouse secondary antibody. Confocal microscopy images were acquired with an ×65 oil lens. Data are representative of ≥3 different experiments.

Article Snippet: Following incubation (40 min at 4°C) with a mouse monoclonal anti-human FPR2 antibody (clone: 6C7-3; kind gift of Dr. D Anderson, Astra Zeneca, Macclesfield, UK) or an isotype control (Serotec, Oxford, UK), cells were washed twice with PBC and incubated for another 30 min at 4°C with an anti-mouse FITC-conjugated secondary antibody (clone: Star 9B; Serotec).

Techniques: Transfection, Labeling, Staining, Confocal Microscopy

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: FPR2/ALX receptor expression and internalization are critical for lipoxin A 4 and annexin-derived peptide-stimulated phagocytosis

doi: 10.1096/fj.10-159913

Figure Lengend Snippet: Cryosectioning and immunogold labeling of FPR2/ALX. Hela cells transiently transfected with HA-tagged FPRL1/ALX were treated with vehicle (EtOH; A) or LXA4 (1 nM) for 5 min (B), 15 min (C), and 30 min (D). Cells were fixed with paraformaldehyde/glutaraldehyde and cryosectioned (60-nm sections). Sections were incubated with anti-HA antibody, followed by an anti-mouse 10-nm gold-conjugated antibody, and contrasted by incubation in uranyl acetate in methyl cellulose. View: ×65,000 (A, D); ×100,000 (B, C).

Article Snippet: Following incubation (40 min at 4°C) with a mouse monoclonal anti-human FPR2 antibody (clone: 6C7-3; kind gift of Dr. D Anderson, Astra Zeneca, Macclesfield, UK) or an isotype control (Serotec, Oxford, UK), cells were washed twice with PBC and incubated for another 30 min at 4°C with an anti-mouse FITC-conjugated secondary antibody (clone: Star 9B; Serotec).

Techniques: Labeling, Transfection, Incubation

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: FPR2/ALX receptor expression and internalization are critical for lipoxin A 4 and annexin-derived peptide-stimulated phagocytosis

doi: 10.1096/fj.10-159913

Figure Lengend Snippet: Quantification of LXA4- and Ac2–26-induced internalization of FPR2/ALX by ELISA and FACS. A, B) Hela cells transiently expressing HA-tagged FPR2/ALX were stimulated for the indicated times with 1 nM LXA4 (A) or 30 μM Ac2–26 (B). The loss of cell surface HA expression, index of receptor internalization, was measured by ELISA, as described in Materials and Methods. Data represent means ±se; n = 4–6 *P < 0.05 vs. vehicle. C) Isolated human PMNs or HEK cells transfected with the FPR2 receptor were incubated with vehicle, 10 μM Ac2–26, or 1 nM LXA4 for the indicated times. At the end of each point, further receptor movement was inhibited. Following incubation with a mouse monoclonal anti-human FPR2 antibody or an isotype, control cells were washed and incubated for a further 30 min at 4°C with an anti-mouse FITC-conjugated secondary antibody. At least 10,000 events were analyzed using a FACSCalibur flow cytometer and CellQuest software. Surface protein expression was recorded as MFI units measured in the FL1 green channel. Data are means ± se from ≥4 independent experiments for both PMNs and HEK cells.

Article Snippet: Following incubation (40 min at 4°C) with a mouse monoclonal anti-human FPR2 antibody (clone: 6C7-3; kind gift of Dr. D Anderson, Astra Zeneca, Macclesfield, UK) or an isotype control (Serotec, Oxford, UK), cells were washed twice with PBC and incubated for another 30 min at 4°C with an anti-mouse FITC-conjugated secondary antibody (clone: Star 9B; Serotec).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Isolation, Transfection, Incubation, Control, Flow Cytometry, Software

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: FPR2/ALX receptor expression and internalization are critical for lipoxin A 4 and annexin-derived peptide-stimulated phagocytosis

doi: 10.1096/fj.10-159913

Figure Lengend Snippet: Protein kinase C is involved in the LXA4-induced internalization of FPR2/ALX. Hela cells transiently expressing HA-tagged FPR2/ALX were pretreated for 30 min with vehicle (Veh), BisI (10 μM), wortmannin (100 nM), or genistein (100 μM), and then stimulated with LXA4 (1 nM) for 15 min, and loss of cell surface receptors was assessed by ELISA (n=3). *P < 0.05 vs. vehicle.

Article Snippet: Following incubation (40 min at 4°C) with a mouse monoclonal anti-human FPR2 antibody (clone: 6C7-3; kind gift of Dr. D Anderson, Astra Zeneca, Macclesfield, UK) or an isotype control (Serotec, Oxford, UK), cells were washed twice with PBC and incubated for another 30 min at 4°C with an anti-mouse FITC-conjugated secondary antibody (clone: Star 9B; Serotec).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: FPR2/ALX receptor expression and internalization are critical for lipoxin A 4 and annexin-derived peptide-stimulated phagocytosis

doi: 10.1096/fj.10-159913

Figure Lengend Snippet: Disruption of lipid raft affected FPR2/ALX internalization. Hela cells transiently expressing HA-tagged FPR2/ALX were pretreated with sucrose (0.45 M) or filipin (5 μg/ml) for 30 min prior to treatment with LXA4 (1 nM) for 15 min. Loss of cell surface receptors was assessed by ELISA (n=3).

Article Snippet: Following incubation (40 min at 4°C) with a mouse monoclonal anti-human FPR2 antibody (clone: 6C7-3; kind gift of Dr. D Anderson, Astra Zeneca, Macclesfield, UK) or an isotype control (Serotec, Oxford, UK), cells were washed twice with PBC and incubated for another 30 min at 4°C with an anti-mouse FITC-conjugated secondary antibody (clone: Star 9B; Serotec).

Techniques: Disruption, Expressing, Enzyme-linked Immunosorbent Assay

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: FPR2/ALX receptor expression and internalization are critical for lipoxin A 4 and annexin-derived peptide-stimulated phagocytosis

doi: 10.1096/fj.10-159913

Figure Lengend Snippet: BMDMs from Fpr2−/− mice show defective phagocytosis of apoptotic PMNs. BMDMs isolated from WT or Fpr2−/− mice were exposed to vehicle, LXA4 (1 nM) or Ac2–26 (10–30 μM) before incubation with human apoptotic PMNs. Phagocytosis was allowed to proceed for 30 min. Phagocytosis was quantified my microscopy after staining for myeloperoxidase as described in Materials and Methods. Data are expressed as mean ± se percentage of phagocytosis (n=4). *P < 0.05, **P < 0.001, #P < 0.05 vs. vehicle.

Article Snippet: Following incubation (40 min at 4°C) with a mouse monoclonal anti-human FPR2 antibody (clone: 6C7-3; kind gift of Dr. D Anderson, Astra Zeneca, Macclesfield, UK) or an isotype control (Serotec, Oxford, UK), cells were washed twice with PBC and incubated for another 30 min at 4°C with an anti-mouse FITC-conjugated secondary antibody (clone: Star 9B; Serotec).

Techniques: Isolation, Incubation, Microscopy, Staining