Journal: Journal of Neuroscience

Article Title: The 2 Subunit of GABAA Receptors Is a Substrate for Palmitoylation by GODZ

doi: 10.1523/jneurosci.1037-04.2004

Figure Lengend Snippet: Figure 2. GFP-tagged GODZ partly colocalizes and interacts with 2 subunit-containing GABAA receptors in transfected HEK 293T cells. A–C, HEK 293T cells were transfected with the (9E10)2 subunit alone (A) or together with GFP-GODZ (B, C) and fixed and permeabilized. The 2 subunit was stained with mAb 9E10 and secondary antibody (red); colocalization with GFP-GODZ (green) is shown in yellow. Note the reduced expression of the 2 subunit in the plasma membrane and the increased intracellular localization of the 2 subunit after expres- sion of GFP-GODZ. D–L, HEK 293T cells were cotransfected with 23 (9E10)2 receptors and GFP-GODZandprocessedasabove(D–I)orincubatedwhilealiveandthereceptorsaggregated withmAb9E10andsecondaryantibody(J–L).The2subunitvisualizedwithmAb9E10(D–L) is shown in red, GFP-GODZ (E, F, H, I, K, L) is shown in green, and the merged panels (F, I) illustrate colocalization (yellow) or the lack thereof (L). Note the variable subcellular localiza- tion of GFP-GODZ. M–R, HEK 293T cells were cotransfected with GFP-GODZC and either 23 (9E10)2 (M–O) or 23 receptors (P–R). The cells were fixed, permeabilized, and processed for immunofluorescence using mAb 9E10 or anti-2 antiserum to visualize the 2 (M, O) and 2 subunit (P, R), respectively, in red. GFP-GODZC is shown in green. Note the localizationofGFP-GODZCtointracellularcompartments(N,Q),accompaniedbyintracellular accumulationofthe2subunit(M,O).Incontrast,nosuchintracellularlocalizationisseenfor the2subunitwhen23subunitsarecotransfectedwithGFP-GODZCintheabsenceofthe 2 subunit (P–R). Images represent single optical sections. Arrows indicate colocalization. Scale bar, 5 m.

Article Snippet: HEK 293T cells were obtained from ATCC (Manassas, VA) and maintained in DMEM (Invitrogen) supplemented with 10% fetal calf serum at 37°C in 5% CO2.

Techniques: Transfection, Staining, Expressing, Clinical Proteomics, Membrane, Immunofluorescence

Journal: Journal of Neuroscience

Article Title: The 2 Subunit of GABAA Receptors Is a Substrate for Palmitoylation by GODZ

doi: 10.1523/jneurosci.1037-04.2004

Figure Lengend Snippet: Figure3. The2subunitandGODZfromacomplexintransfectedHEK293Tcells.Todirectly testwhetherGABAAreceptorsandGODZcanexistinacomplexinmammaliancells,weimmu- noprecipitated the 2 subunit from extracts of HEK 293T cells cotransfected with vectors en- codingthe2subunitandFLAG-GODZ.A,Westernblotanalysesof2subunitimmunoprecipi- tates revealed efficient copurification of GODZ. B, Conversely, immunopurification of FLAG- GODZ from the same extracts revealed coimmunoprecipitation of the 2 subunit, confirming thatthetwoproteinscanexistinacomplexwhenoverexpressedinHEK293Tcells.Notethatthe weak unspecific band seen in both the FLAG immunoprecipitate and the IgG control lane (B, bottom panel) runs at 50 kDa just above the 2 subunit. Results shown are representative of three (A) and five (B) successful experiments, respectively.

Article Snippet: HEK 293T cells were obtained from ATCC (Manassas, VA) and maintained in DMEM (Invitrogen) supplemented with 10% fetal calf serum at 37°C in 5% CO2.

Techniques: Copurification, Immu-Puri, Control

Journal: Journal of Neuroscience

Article Title: The 2 Subunit of GABAA Receptors Is a Substrate for Palmitoylation by GODZ

doi: 10.1523/jneurosci.1037-04.2004

Figure Lengend Snippet: Figure 5. GODZ antiserum recognizes recombinant and native GODZ in vitro and in vivo. A, ImmunoblotofextractspreparedfromHEK293TcellstransfectedwithFLAG-GODZ(lanes1,2), untransfected HEK 293T cells (lane 3), and brain membranes (lane 4) were visualized using FLAG (lane 1) or GODZ (lanes 2–4), respectively. The arrows at 31 kDa indicate the mobilityofendogenousGODZandFLAG-GODZ.B,Immunoblotofextractspreparedfrombrain membranes(lanes1,2)developedwitheitherGODZantiserum(lane1)orGODZantiserumthat waspreadsorbedwiththeimmunizingpeptideantigen(lane2).C,FLAG-GODZwastransfected intoHEK293TcellsandstainedwithanantibodydirectedagainsttheFLAGepitopetag(green) or with the GODZ antiserum (red). Results shown are representative of at least three similar experiments each.

Article Snippet: HEK 293T cells were obtained from ATCC (Manassas, VA) and maintained in DMEM (Invitrogen) supplemented with 10% fetal calf serum at 37°C in 5% CO2.

Techniques: Recombinant, In Vitro, In Vivo

Journal: Journal of Neuroscience

Article Title: The 2 Subunit of GABAA Receptors Is a Substrate for Palmitoylation by GODZ

doi: 10.1523/jneurosci.1037-04.2004

Figure Lengend Snippet: Figure7. The2subunitcytoplasmicloopisasubstrateforpalmitoylationbyGODZ.A–C,Recombinant (9E10)2subunitand FLAG-GODZwerecotransfectedandexpressedinHEK293Tcells,metabolicallylabeledwith[ 3H]palmiticacidfor4–5hr,immu- noprecipitated with anti-2 antiserum or IgG control serum, and subjected to SDS-PAGE and fluorography (A), Western blot analysis using mAb 9E10 (B), or hydroxylamine treatment before fluorography (C). D, Recombinant 1(9E10)2 receptors together with FLAG-GODZ (lanes 1, 3) or alone (lanes 2, 4) were cotransfected and expressed in HEK 293T cells, metabolically labeled with [ 3H]palmitic acid for 4–5 hr, immunoprecipitated with anti-2 antiserum or IgG control serum, subjected to SDS-PAGEandfluorography(toppanel),orvisualizedbyWesternblotwithmAb9E10(bottompanel).E,F,Recombinant (9E10)2 and either GFP-GODZ (lane 1) or FLAG-GODZ (lane 2) were cotransfected and expressed in HEK 293T cells, metabolically labeled with [ 3H]palmitic acid for 4–5 hr, and immunoprecipitated with anti-2 antiserum. The sample was divided, subjected to SDS-PAGE, and processed for fluorography (E) and Western blot analysis using GODZ antiserum (F), respectively. The faster- migrating GFP-GODZ species is likely to represent a degradation product of the full-length product. G, FLAG-GODZ was cotrans- fected with recombinant 12 subunits and (9E10)2 (lane 1), empty vector (pRK5, lane 2), or a (9E10)2/2 chimeric construct (lane 3; see Materials and Methods) into HEK 293T cells. The cells were metabolically labeled with [ 3H]palmitic acid, immuno- precipitated with anti-2 antiserum, and fractions of the sample were processed for fluorography (top panel) and Western blot (bottom panel) using an anti-myc antiserum, respectively. H, Cultured cortical neurons were metabolically labeled with [ 3H]palmitic acid. GABAA receptors were immunoprecipitated with anti-1 antiserum or IgG control serum and processed for fluorography (top panel) and Western blot (bottom panel) using anti-2 antiserum as in G. Results shown are representative of two to three similar experiments each.

Article Snippet: HEK 293T cells were obtained from ATCC (Manassas, VA) and maintained in DMEM (Invitrogen) supplemented with 10% fetal calf serum at 37°C in 5% CO2.

Techniques: Recombinant, Control, SDS Page, Western Blot, Metabolic Labelling, Labeling, Immunoprecipitation, Plasmid Preparation, Construct, Cell Culture

Journal: Journal of Biological Chemistry

Article Title: Internalization of Exogenously Added Memapsin 2 (β-Secretase) Ectodomain by Cells Is Mediated by Amyloid Precursor Protein

doi: 10.1074/jbc.m402130200

Figure Lengend Snippet: FIG. 3. M2ED internalization and the effect of PI-PLC analyzed by flow cytometry. HEK293 cells stably transfected with APPsw were incubated in the presence or absence of 2.5 units/ml PI-PLC at either 37 °C (A) or on ice (B), for 2 h, followed by an additional 2-h incubation with either 0 or 42 g of Alexa 488-M2ED at the indicated temperatures. Cells were then washed extensively with 0.05% Tween 20/PBS and resuspended in 0.5 ml of ice-cold PBS for flow cytometric analysis (FACSCalibur). Data are representative of two independent experiments.

Article Snippet: Human embryonic kidney (HEK293) and HeLa cells were obtained from American Type Culture Collection and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) containing 10% heat-inactivated fetal bovine serum (Invitrogen) and 2% penicillin/streptomycin (10,000 IU/ml and 10,000 g/ml, Cellgro), designated as complete DMEM.

Techniques: Flow Cytometry, Stable Transfection, Transfection, Incubation

Journal: Journal of Biological Chemistry

Article Title: Internalization of Exogenously Added Memapsin 2 (β-Secretase) Ectodomain by Cells Is Mediated by Amyloid Precursor Protein

doi: 10.1074/jbc.m402130200

Figure Lengend Snippet: FIG. 6. Internalized M2ED supported the production of A, which is influenced by GPI-anchored proteins at the cell surface. A, confocal microscopic images of cells incubated with Alexa 488-M2ED with or without PI-PLC. HeLa/APPsw cells were initially incubated with 2.5 units/ml of PI-PLC in Opti-MEM at 37 °C for 2 h, followed by an additional 2-h incubation of 45 g/ml Alexa 488-M2ED. B, quantitative analysis of cell-associated fluorescence as shown in A. The ordinate of the graph expresses the signal in arbitrary fluorescent units. Statistical significance (asterisk) was indicated by a p value of 0.00076. C, the production of membrane-associated C-terminal fragment (CTF), soluble APP (sAPP), and A as shown by autoradiogram from SDS-PAGE. HEK 293/APPsw cells were metabolically radiolabeled with [35S]methionine then incubated with the combinations of M2ED and/or glycosylphosphatidylinositol-specific phospholipase C (PI-PLC) as indicated. Significant increases of sAPP, CTF, and A bands were seen in the M2ED-added cells (center lane) compared with the control (without M2ED, left lane). Incubation of cells with 1.0 unit/ml PI-PLC for 2 h reduced sAPP, CTF, and A production to a level close to that of the control (right lane). Data are representative of three independent experiments.

Article Snippet: Human embryonic kidney (HEK293) and HeLa cells were obtained from American Type Culture Collection and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) containing 10% heat-inactivated fetal bovine serum (Invitrogen) and 2% penicillin/streptomycin (10,000 IU/ml and 10,000 g/ml, Cellgro), designated as complete DMEM.

Techniques: Incubation, Fluorescence, Membrane, SDS Page, Metabolic Labelling, Control

Journal: Journal of Biological Chemistry

Article Title: Internalization of Exogenously Added Memapsin 2 (β-Secretase) Ectodomain by Cells Is Mediated by Amyloid Precursor Protein

doi: 10.1074/jbc.m402130200

Figure Lengend Snippet: FIG. 9. APP and full-length memapsin 2 immunoprecipitated together by either APP antibody or memapsin 2 antibody. A, parental HEK293 cells (marked 293) or the same cells transfected with memapsin 2 and APP Swedish mutant (marked 293/M2/APPsw) were treated with or without 2.0 units/ml of PI-PLC for 2 h. Cell lysates were then separately immunoprecipitated with anti-memapsin 2 antibody and analyzed by immunoblotting with anti-APP antibody. B, cell ly- sates were immunoprecipitated with anti-APP antibody and Western blotted with anti-memapsin 2 antibody. Data are representative of five independent experiments.

Article Snippet: Human embryonic kidney (HEK293) and HeLa cells were obtained from American Type Culture Collection and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) containing 10% heat-inactivated fetal bovine serum (Invitrogen) and 2% penicillin/streptomycin (10,000 IU/ml and 10,000 g/ml, Cellgro), designated as complete DMEM.

Techniques: Immunoprecipitation, Transfection, Mutagenesis, Western Blot

Journal:

Article Title: Cyclophilin C-associated protein: A normal secreted glycoprotein that down-modulates endotoxin and proinflammatory responses in vivo

doi:

Figure Lengend Snippet: Biosynthesis and secretion of CyCAP. (A) Pulse–chase analysis of CyCAP in MM55 kidney cells. Cells were pulsed for 10 min with [35S]cysteine and [35S]methionine and chased for the indicated times. CyCAP was immunoprecipitated from cell lysates and media with polyclonal rat serum against CyCAP. Control precipitations with normal rat serum were negative (data not shown). Proteins were resolved by using SDS/7.5% PAGE. Closed arrows represent positions of molecular-mass standards (Rainbow, Amersham Pharmacia). (B) CyCAP secretion from thioglycolate-elicited peritoneal macrophages. PECs were metabolically labeled overnight, and cypC-GST-glutathione agarose was used as an affinity reagent for CyCAP in the absence of CsA (lanes 3 and 5) or in the presence of CsA (lanes 4 and 6). Antibodies to macrophage markers Mac-1 and Mac-2 were used as positive controls (lanes 1 and 2).

Article Snippet: MM55 mouse kidney cells (ATCC CRL 6436) and the HT-29 human colon carcinoma cell line (ATCC HTB 38) were grown in DMEM with 10% fetal calf serum, 2 mM glutamine, and penicillin/streptomycin.

Techniques: Pulse Chase, Immunoprecipitation, Metabolic Labelling, Labeling

Journal: Nucleic Acids Research

Article Title: SECIS elements in the coding regions of selenoprotein transcripts are functional in higher eukaryotes

doi: 10.1093/nar/gkl1060

Figure Lengend Snippet: Eukaryotic SECIS elements located in coding regions are functional. ( A ) Western blot analysis of HEK293 cells transfected with GPx1 constructs containing functional in-frame SECIS elements. The indicated constructs were generated by either deleting the stop codon TAA or replacing the GPx1 3′-UTR with the fowlpox virus SECIS element thereby generating a GPx1-FPV fusion protein. ( B ) The same samples as shown in A were metabolically labeled with 75 Se and exposed to a PhosphorImager cassette. Location of selenoproteins expressed with the help of coding region SECIS elements is shown by arrows on the right. ( C ) Western blot analysis of HEK293 cells transfected with GPx4 constructs containing in-frame SECIS elements. GPx4-based constructs were generated by replacing non-homologous sequences with the fowlpox SECIS element thereby generating a GPx4-FPV fusion protein. Lane designations are as follows: Mock, control transfection; WT GPx1, wild-type GPx1 gene; GPx1 ΔATGA, mouse GPx1 gene with disrupted SBP2-binding region; GPx1 ΔTAA, mouse GPx1 gene with its natural stop codon deleted (allowing the ORF to extend beyond the SECIS element which becomes part of the ORF); GPx1 FPV SECIS, the fowlpox SECIS element replacing the 3′-UTR region of the mouse GPx1 gene; GPx1 FPV SECIS ΔATGA, the fowlpox virus SECIS was fused in frame with GPx1 and its SBP2-binding site was disrupted; WT GPx4, cells were transfected with wild-type mouse GPx4 cDNA; GPx4 ΔATGA, the SBP2-binding site in the wild-type GPx4 cDNA was disrupted; GPx4 FPV SECIS, the fowlpox virus SECIS element was fused in frame with GPx4 lacking its own 3′-UTR; GPx4 FPV SECIS ΔATGA, the fowlpox virus SECIS fused to GPx4 was mutated to disrupt the SBP2-binding site.

Article Snippet: Mouse hepatoma cell line Hepa 1–6 and human embryonic kidney HEK293 cells (ATCC, Manassas, VA) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin and 100 U/ml streptomycin.

Techniques: Functional Assay, Western Blot, Transfection, Construct, Generated, Virus, Metabolic Labelling, Labeling, Control, Binding Assay

Journal: Nucleic Acids Research

Article Title: SECIS elements in the coding regions of selenoprotein transcripts are functional in higher eukaryotes

doi: 10.1093/nar/gkl1060

Figure Lengend Snippet: Expression of wild-type fowlpox virus GPx4 in mammalian cell lines. ( A ) Western blot analysis of mouse hepatoma cell line Hepa1-6 transfected with wild-type fowlpox virus GPx4 construct (WT FPV GPx) or the corresponding construct in which the SBP2-binding site was disrupted (WT FPV GPx ΔATGA). ( B ) Transfection of HEK293 with wild-type fowlpox virus GPx4 (FPV GPx), its mutants lacking SBP2-binding site (WT FPV GPx ΔATGA) or the conserved AA in the apical loop (WT FPV GPx ΔAA), and the corresponding Sec-to-Cys mutants.

Article Snippet: Mouse hepatoma cell line Hepa 1–6 and human embryonic kidney HEK293 cells (ATCC, Manassas, VA) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin and 100 U/ml streptomycin.

Techniques: Expressing, Virus, Western Blot, Transfection, Construct, Binding Assay

Journal: Nucleic Acids Research

Article Title: SECIS elements in the coding regions of selenoprotein transcripts are functional in higher eukaryotes

doi: 10.1093/nar/gkl1060

Figure Lengend Snippet: Expression of GPx1 containing a designed in-frame SECIS element. ( A ) Hepa1-6 cells were transfected with WT GPx1 and different in-frame SECIS element containing constructs. The construct GPx1 2.IFS ΔATGA contains an additional modification in the stem-region as shown in . To disrupt the function of the natural SECIS element, mutations were made in either the SBP2-binding region or the apical loop. ( B ) Transfection of HEK293 cells. No expression was detectable for GPx4 containing a designed in-frame SECIS element. Note the low expression level of the Cys mutant with the designed in-frame SECIS element compared with GPx1. Expressed proteins were detected by western blot analysis using antibodies specific for V5 tag.

Article Snippet: Mouse hepatoma cell line Hepa 1–6 and human embryonic kidney HEK293 cells (ATCC, Manassas, VA) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin and 100 U/ml streptomycin.

Techniques: Expressing, Transfection, Construct, Modification, Binding Assay, Mutagenesis, Western Blot

Journal:

Article Title: VP1, the Putative RNA-Dependent RNA Polymerase of Infectious Bursal Disease Virus, Forms Complexes with the Capsid Protein VP3, Leading to Efficient Encapsidation into Virus-Like Particles

doi:

Figure Lengend Snippet: Characterization of the expression of IBDV VP1 in cells infected with the rVV VT7/VP1. (A) BSC-1 cells infected with VT7/VP1 or the parental virus VT7LacOI, either treated (+) or untreated (−) with the inducer IPTG, were metabolically labelled with [35S]methionine. CEF mock-infected (Mock) or infected with IBDV (IBDV) were metabolically labelled with [35S]methionine at 48 h p.i. Protein samples from the different cultures were analyzed by SDS-PAGE. After electrophoresis, the gel was electroblotted onto nitrocellulose. The filter was air-dried and subjected to autoradiography. The positions of molecular weight markers are indicated (Mw). The positions of bands corresponding to the VP1 polypeptide are indicated by open arrowheads. (B) Western blot analysis of proteins expressed in cells infected with the rVV VT7/VP1 and CEF infected with IBDV. To determine the position of the VP1 polypeptide, after autoradiography, the nitrocellulose filter was rehydrated and incubated with a rabbit anti-VP1 antisera followed by the addition of horseradish peroxidase-conjugated goat anti-rabbit Ig. The signal was detected by ECL.

Article Snippet: The rVVs VT7/POLY, VT7/VP3, and VT7/VP2 have been previously described ( 7 , 8 ). rVVs were propagated and titrated in African green monkey kidney epithelial BSC-1 cells (American Type Culture Collection) as described previously ( 5 ).

Techniques: Expressing, Infection, Metabolic Labelling, SDS Page, Electrophoresis, Autoradiography, Molecular Weight, Western Blot, Incubation

Journal: Frontiers in Surgery

Article Title: Intraoperative Fluorescence Imaging for Personalized Brain Tumor Resection: Current State and Future Directions

doi: 10.3389/fsurg.2016.00055

Figure Lengend Snippet: Summary of published preclinical and early clinical data on probes and imaging equipment for potential personalized fluorescence-guided brain tumor surgery .

Article Snippet: CLR1502 ( ) , 760 nm , 778 nm , IVIS Spectrum system (PerkinElmer, Inc.) Fluobeam 800 (Fluoptics, Grenoble, France) Leica OH4 intraoperative microscope with FL800 attachment (Leica Microsystems, Bannockburn, IL, USA) , Xenograft mouse model (U251 human glioblastoma, 22T, 22CSC, 33CSC, 105CSC patient derived glioblastoma) , Tumor-to-brain fluorescence ratio superior to 5-ALA , Tumor must be visualized on separate monitor , IV, >4 days.

Techniques: Imaging, Fluorescence, In Vivo, Inverted Microscopy, Ex Vivo, Labeling, Concentration Assay, Derivative Assay, In Vitro, Expressing, Microscopy, Produced, Injection, Cell Culture, Magnetic Resonance Imaging, Staining, Construct, Binding Assay, In Vivo Imaging, Activity Assay, Modification, Laser-Scanning Microscopy, Incubation, Metabolic Labelling, Spectroscopy

Journal: PLoS Pathogens

Article Title: Varicellovirus UL49.5 Proteins Differentially Affect the Function of the Transporter Associated with Antigen Processing, TAP

doi: 10.1371/journal.ppat.1000080

Figure Lengend Snippet: (A) MHC class I expression in cells from the relevant host species stably expressing the UL49.5 homologs of BHV-1, PRV, EHV-1, EHV-4, CHV and VZV. Cells of bovine (MDBK), porcine (PK15), equine (E.derm), canine (MDCK), and human origin (MJS) were transduced with retroviruses expressing the respective UL49.5 proteins. Surface MHC class I molecules were stained with specific antibodies and analyzed using flow cytometry. Shown are the MHC class I levels on cells without UL49.5 (thin line) and with UL49.5 (boldface line); dashed line, goat anti-mouse phycoerythrin control. (B) TAP-dependent peptide transport is inhibited in cells from the relevant host species expressing BHV-1, PRV, EHV-1, EHV-4 and CHV UL49.5, but not in MJS cells expressing VZV UL49.5. Peptide transport was assessed in the presence and absence of ATP (black and open bars, respectively). Peptide transport is expressed as percentage of translocation, relative to the translocation observed in control cells (defined as 100%). The standard deviations are represented by the error bars. A difference at p<0.05 was considered significant.

Article Snippet: The human melanoma cell line Mel JuSo (MJS), MJS TAP1-GFP and Madin-Darby bovine kidney (MDBK) cells (American Type Culture Collection, ATCC) were maintained in RPMI-1640 medium; GP2-293 cells, porcine kidney (PK15) cells, the embryonic bovine trachea (EBTr) cell line, Madin-Darby canine kidney I (MDCK I) cells, and the equine epithelial cell line E.derm were maintained in DMEM medium.

Techniques: Expressing, Stable Transfection, Transduction, Staining, Flow Cytometry, Control, Translocation Assay

Journal: PLoS Pathogens

Article Title: Varicellovirus UL49.5 Proteins Differentially Affect the Function of the Transporter Associated with Antigen Processing, TAP

doi: 10.1371/journal.ppat.1000080

Figure Lengend Snippet: Bovine cells (MDBK), porcine cells (PK15), and equine cells (E.derm) were infected with wild type BHV-1, PRV, or EHV-1, respectively, or with the corresponding UL49.5-negative recombinant viruses. In all experiments, mock-treated (uninfected) cells from the relevant host species were used as a control. Peptide transport was assessed at 5 hrs post-infection in the presence and absence of ATP (black and open bars, respectively). The data are expressed as percentage of translocation, relative to the translocation observed in control cells (defined as 100%).

Article Snippet: The human melanoma cell line Mel JuSo (MJS), MJS TAP1-GFP and Madin-Darby bovine kidney (MDBK) cells (American Type Culture Collection, ATCC) were maintained in RPMI-1640 medium; GP2-293 cells, porcine kidney (PK15) cells, the embryonic bovine trachea (EBTr) cell line, Madin-Darby canine kidney I (MDCK I) cells, and the equine epithelial cell line E.derm were maintained in DMEM medium.

Techniques: Infection, Recombinant, Control, Translocation Assay

Journal: PLoS Pathogens

Article Title: Varicellovirus UL49.5 Proteins Differentially Affect the Function of the Transporter Associated with Antigen Processing, TAP

doi: 10.1371/journal.ppat.1000080

Figure Lengend Snippet: MDBK cells were mock-infected or infected with wild type BHV-1 or BHV-1 expressing UL49.5Δtail for 12 hours. The levels of bovine TAP1 and, as a control, α-tubulin were assessed in lysates of infected cells by immunoblotting (IB). BHV-1 UL49.5 was immunoprecipitated from lysates of metabolically labeled cells (IP).

Article Snippet: The human melanoma cell line Mel JuSo (MJS), MJS TAP1-GFP and Madin-Darby bovine kidney (MDBK) cells (American Type Culture Collection, ATCC) were maintained in RPMI-1640 medium; GP2-293 cells, porcine kidney (PK15) cells, the embryonic bovine trachea (EBTr) cell line, Madin-Darby canine kidney I (MDCK I) cells, and the equine epithelial cell line E.derm were maintained in DMEM medium.

Techniques: Infection, Expressing, Control, Western Blot, Immunoprecipitation, Metabolic Labelling, Labeling

Journal: Nature

Article Title: Transferrin receptor 1 is a cellular receptor for New World haemorrhagic fever arenaviruses

doi: 10.1038/nature05539

Figure Lengend Snippet: a , Schematic representation of MACV glycoprotein precursor (GPC), GP1-Ig and GP1Δ-Ig. Locations of the signal peptide (SP), the GP1 and GP2 domains and the transmembrane domain (TM) are indicated. b , Vero cells analysed by flow cytometry with the use of the indicated concentrations of purified GP1-Ig (circles) or GP1Δ-Ig (squares). m.f.i., mean fluorescence intensity. Error bars indicate standard deviation. c , Vero cells were metabolically labelled, incubated with GP1Δ-Ig or the indicated control Ig-fusion proteins, lysed, immunoprecipitated, and analysed by SDS–PAGE under reducing (left) or non-reducing (right) conditions. d , Binding of anti-human TfR1 antibody (left) and MACV GP1-Ig (right) to CHO (light grey) or CHO-hTfR1 (dark grey) cells analysed by flow cytometry. The unfilled curve in the right-hand panel is secondary antibody alone.

Article Snippet: Vero cells (African green monkey kidney epithelial; CCL-81; ATCC), 293T cells (human kidney epithelial; CRL-11268; ATCC), HeLa 229 cells (human cervical epithelial; CCL-2.1; ATCC) and 3T3-Swiss cells (mouse embryo fibroblast; CCL-92; ATCC) were grown in DMEM medium; LLC-MK2 cells (rhesus monkey kidney epithelial; CCL-7; ATCC) in medium 199; BHK-21 cells (Chinese hamster kidney fibroblast; CCL-10; ATCC) and EJG cells (bovine capillary endothelial; CRL-8659; ATCC) in minimal essential medium (Eagle); SLK cells (human endothelial; NIH AIDS Research & Reference Regent Program) in RPMI medium; CHO cells (Chinese hamster ovary epithelial) in F12 medium; CHO-hTfR1 cells (a gift from T. McGraw) in F12 medium containing 400 μg ml -1 G418.

Techniques: Flow Cytometry, Purification, Fluorescence, Standard Deviation, Metabolic Labelling, Incubation, Control, Immunoprecipitation, SDS Page, Binding Assay

Journal: Molecular microbiology

Article Title: Sphingomyelin, glycosphingolipids and ceramide signalling in cells exposed to P-fimbriated Escherichia coli.

doi: 10.1046/j.1365-2958.1998.01017.x

Figure Lengend Snippet: Fig. 1. Release of ceramide after stimulation of A498 cells with 108 cfu ml¹1 E. coli AD110 (P-fimbriated wild type), E. coli HB101 (pPIL110-35) (P-fimbriated recombinant) or non-E. coli HB101 (non-fimbriated recipient). E. coli AAEC (non-fimbriated recipient), E. coli AAEC (pPKL4) (type 1 fimbriated recombinant). Lipids were extracted and subjected to mild alkaline hydrolysis. The levels of free ceramide were determined by the DAG kinase assay.

Article Snippet: Cell activation assays The human epithelial cell line A498 (ATCC HTB44, human Q 1998 Blackwell Science Ltd, Molecular Microbiology, 29, 1297–1306 kidney carcinoma) was grown in RPMI-1640 supplemented with 5% FCS, 1 mM sodium pyruvate, 1 mM non-essential amino acids, gentamicin (50 mg ml11) and 2 mM L-glutamine.

Techniques: Recombinant, Kinase Assay

Journal: Molecular microbiology

Article Title: Sphingomyelin, glycosphingolipids and ceramide signalling in cells exposed to P-fimbriated Escherichia coli.

doi: 10.1046/j.1365-2958.1998.01017.x

Figure Lengend Snippet: Fig. 3. Release of ceramide by A498 cells stimulated with agonists known to activate the sphingomyelin cycle. Cells were exposed to exogenous SMase (1 U ml¹1), TNF-a (10 ng ml¹1), IL-1b (10 ng ml¹1) or to medium for 10 or 20 min. Lipids were extracted and subjected to mild alkaline hydrolysis. The levels of free ceramide were determined by the DAG kinase assay.

Article Snippet: Cell activation assays The human epithelial cell line A498 (ATCC HTB44, human Q 1998 Blackwell Science Ltd, Molecular Microbiology, 29, 1297–1306 kidney carcinoma) was grown in RPMI-1640 supplemented with 5% FCS, 1 mM sodium pyruvate, 1 mM non-essential amino acids, gentamicin (50 mg ml11) and 2 mM L-glutamine.

Techniques: Kinase Assay

Journal: Molecular microbiology

Article Title: Sphingomyelin, glycosphingolipids and ceramide signalling in cells exposed to P-fimbriated Escherichia coli.

doi: 10.1046/j.1365-2958.1998.01017.x

Figure Lengend Snippet: Fig. 2. P fimbriae augment epithelial cytokine responses to E. coli. A498 cells grown to confluency in 24-well plates were stimulated with E. coli AD110 (W), E. coli HB101(pPIL110-35) (S), non-E. coli HB101 (D), E. coli AAEC (B), E. coli AAEC (pPKL4) (l) or medium (A). Supernatants were collected at 0, 2, 6 and 24 h, and IL-6 levels were determined by ELISA.

Article Snippet: Cell activation assays The human epithelial cell line A498 (ATCC HTB44, human Q 1998 Blackwell Science Ltd, Molecular Microbiology, 29, 1297–1306 kidney carcinoma) was grown in RPMI-1640 supplemented with 5% FCS, 1 mM sodium pyruvate, 1 mM non-essential amino acids, gentamicin (50 mg ml11) and 2 mM L-glutamine.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Molecular microbiology

Article Title: Sphingomyelin, glycosphingolipids and ceramide signalling in cells exposed to P-fimbriated Escherichia coli.

doi: 10.1046/j.1365-2958.1998.01017.x

Figure Lengend Snippet: Fig. 4. Epithelial cytokine responses to IL-1b (10 ng ml¹1) (S), TNF-a (10 ng ml¹1) (W), SMase (1 U ml¹1) (D) or medium (A). A498 cells were grown to confluency in 24-well plates and stimulated with the different agonists. Supernatants were collected at 0, 2, 6 and 24 h, and IL-6 levels were determined by ELISA.

Article Snippet: Cell activation assays The human epithelial cell line A498 (ATCC HTB44, human Q 1998 Blackwell Science Ltd, Molecular Microbiology, 29, 1297–1306 kidney carcinoma) was grown in RPMI-1640 supplemented with 5% FCS, 1 mM sodium pyruvate, 1 mM non-essential amino acids, gentamicin (50 mg ml11) and 2 mM L-glutamine.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Molecular microbiology

Article Title: Sphingomyelin, glycosphingolipids and ceramide signalling in cells exposed to P-fimbriated Escherichia coli.

doi: 10.1046/j.1365-2958.1998.01017.x

Figure Lengend Snippet: Fig. 5. Hydrolysis of sphingomyelin after exposure to the different agonists used in this study. A. Agonists that stimulate the sphingomyelin cycle cause sphingomyelin breakdown. A498 cells were labelled metabolically with [14C]-choline (1 mCi ml¹1) and exposed to TNF-a (10 ng ml¹1), IL-1b (10 ng ml¹1), exogenous SMase (1 U ml¹1) or medium for 20 min. Lipids were extracted and separated by TLC. The breakdown of sphingomyelin was detected as the disappearance of label from the alkali-stable lipid fraction of the cells. [14C]-choline was quantified by image analyser (Table 1). SM (1, 2) forms a double band depending on the hydroxylation of ceramide. (3, origin). B. P-fimbriated E. coli strains do not affect the levels of sphingomyelin in human kidney cells. The cells were stimulated for 20 min with E. coli AD110, E. coli HB101(pPIL110-35), non-E. coli HB101 or medium.

Article Snippet: Cell activation assays The human epithelial cell line A498 (ATCC HTB44, human Q 1998 Blackwell Science Ltd, Molecular Microbiology, 29, 1297–1306 kidney carcinoma) was grown in RPMI-1640 supplemented with 5% FCS, 1 mM sodium pyruvate, 1 mM non-essential amino acids, gentamicin (50 mg ml11) and 2 mM L-glutamine.

Techniques: Metabolic Labelling

Journal: Molecular microbiology

Article Title: Sphingomyelin, glycosphingolipids and ceramide signalling in cells exposed to P-fimbriated Escherichia coli.

doi: 10.1046/j.1365-2958.1998.01017.x

Figure Lengend Snippet: Fig. 6. Receptor activity of A498 cell glycosphingolipids for P-fimbriated E. coli as demonstrated by TLC overlay. Non-acid glycosphingolipids extracted from the A498 cell line (lane 1) and purified globotetraosylceramide (lane 2) were run on a TLC plate and overlaid with radiolabelled P fimbriated E. coli HB101(pPIL110- 35) (A) and non-fimbriated E. coli HB101 (B). E. coli HB101 did not bind to the glycolipids.

Article Snippet: Cell activation assays The human epithelial cell line A498 (ATCC HTB44, human Q 1998 Blackwell Science Ltd, Molecular Microbiology, 29, 1297–1306 kidney carcinoma) was grown in RPMI-1640 supplemented with 5% FCS, 1 mM sodium pyruvate, 1 mM non-essential amino acids, gentamicin (50 mg ml11) and 2 mM L-glutamine.

Techniques: Activity Assay

Journal: Molecular microbiology

Article Title: Sphingomyelin, glycosphingolipids and ceramide signalling in cells exposed to P-fimbriated Escherichia coli.

doi: 10.1046/j.1365-2958.1998.01017.x

Figure Lengend Snippet: Fig. 7. [14C]-galactose glycosphingolipids. A498 cells were prelabelled with [14C]-galactose (1 mCi ml¹1) for 48 h and exposed to bacteria for 20 min. Lipids were extracted, subjected to mild alkaline hydrolysis and separated by TLC (A). The E. coli strains were E. coli AD110 (P- fimbriated wild type), E. coli HB101(pPIL110-35) (P-fimbriated recombinant) and E. coli HB101 (non-fimbriated recipient). The [14C]-galactose content of individual bands was quantified (B) using an image analyser and compared using the Wilcoxon test. Numbers are the means of seven experiments and triplicate samples.

Article Snippet: Cell activation assays The human epithelial cell line A498 (ATCC HTB44, human Q 1998 Blackwell Science Ltd, Molecular Microbiology, 29, 1297–1306 kidney carcinoma) was grown in RPMI-1640 supplemented with 5% FCS, 1 mM sodium pyruvate, 1 mM non-essential amino acids, gentamicin (50 mg ml11) and 2 mM L-glutamine.

Techniques: Bacteria, Recombinant