anti rabbit immune serum  (Cell Signaling Technology Inc)

Journal: PLoS ONE

Article Title: An In Vitro System for Studying Murid Herpesvirus-4 Latency and Reactivation

doi: 10.1371/journal.pone.0011080

Figure Lengend Snippet: a . Schematic diagram showing the insertion in the 5′ untranslated region of ORF50, between MuHV-4 ORFs 48 and 49, of a poly-adenylation site plus a downstream promoter (TRE) with binding sites for doxycycline-dependent transcription regulators. b . Viral DNA from the TRE-50 mutant (T50), an independent mutant (ind), a revertant of each mutant (R.4, R.8) and wild-type MuHV-4 (WT) was digested with Bgl I, Eco RI or Bam HI, electrophoresed and probed with a Bam HI-N fragment as shown in a . The polyA-TRE insertion (705bp) introduces a Bgl I site, such that a wild-type 16476bp band becomes 11040bp + 6141bp, and an Eco RI site, such that a 14937bp wild-type band becomes 13926bp + 1716bp. The 3525bp Eco RI band is unchanged. The insertion makes the 4048bp wild-type Bam HI band 4753bp.

Article Snippet: For specific staining, cells were incubated with rabbit anti-MuHV-4 immune serum (1 h, 4°C), washed x2 in PBS, incubated with fluorescein-conjugated pig anti-rabbit pAb (Dako Cytomation) (1 h, 4°C), washed x2 in PBS and analysed on a FACS Calibur using Cellquest software (BD Biosciences).

Techniques: Binding Assay, Mutagenesis

Journal: PLoS ONE

Article Title: An In Vitro System for Studying Murid Herpesvirus-4 Latency and Reactivation

doi: 10.1371/journal.pone.0011080

Figure Lengend Snippet: a . TET-ON/OFF cells were infected with TRE-50 MuHV-4 (3 p.f.u. /cell) then cloned. Cultures of a representative clone were analyzed for infectious virus by plaque assay after induction with doxycycline (dox, 1 µg/ml). Infectious virus was undetectable without induction. b . Plaque titers after doxycycline treatment were determined for an additional 5 clones. The x axis marks the lower limit of assay sensitivity. Again no infectivity was detected without induction. c . 3 clones were tested for sensivity to doxycycline. Virus titers were determined by plaque assay 48 h after adding doxycycline at the final concentration shown. d . A latently infected TET-ON/OFF cell clone was induced with doxycycline for the time indicated (dotted lines) or left uninduced (dashed lines), then stained with a MuHV-4-immune rabbit serum and analyzed by flow cytometry. In the bottom panel, the cells were infected with wild-type MuHV-4 without doxycycline (filled histogram) or left uninfected. e . A latently infected clone (TET-ON/OFF-TRE-50) or uninfected cells (TET-ON/OFF) were treated or not with doxycycline for the time indicated, then analyzed for MuHV-4 antigen expression by immunoblotting with a MuHV-4-immune rabbit serum. As a positive control, wild-type MuHV-4 (virus +) was added 18 h before analysis. f . Latently infected TET-ON/OFF cell clones were sub-cloned further and then cultured with doxycycline. Sub-clone supernatants were analyzed for infectious virus by incubation with BHK-21 cells for 4 days and then scoring or not for cytopathic effects. Each graph shows the percentage of subclones yielding infectious virus with time. At least 70 sub-clones were analyzed for each latently infected clone.

Article Snippet: For specific staining, cells were incubated with rabbit anti-MuHV-4 immune serum (1 h, 4°C), washed x2 in PBS, incubated with fluorescein-conjugated pig anti-rabbit pAb (Dako Cytomation) (1 h, 4°C), washed x2 in PBS and analysed on a FACS Calibur using Cellquest software (BD Biosciences).

Techniques: Infection, Clone Assay, Plaque Assay, Concentration Assay, Staining, Flow Cytometry, Expressing, Western Blot, Positive Control, Cell Culture, Incubation

Journal: PLoS ONE

Article Title: An In Vitro System for Studying Murid Herpesvirus-4 Latency and Reactivation

doi: 10.1371/journal.pone.0011080

Figure Lengend Snippet: a . TET-ON/OFF cells were infected (0.1 p.f.u. /cell) with wild-type eGFP + or TRE-50 eGFP + MuHV-4, then treated or not with doxycycline (dox, 1 µg/ml). 48 later the cells were fixed, permeabilized and stained for ORF65 capsid expression with mAb MG-12B8. EGFP expression was visualized directly. Nuclei were counterstained with DAPI. b . Latent eGFP + TRE-50 clones were established and reactivation then induced or not with doxycycline. 48 h later the cells were fixed, permeabilized and stained as in a . 2 representative clones (3.4, 10.4) are shown.

Article Snippet: For specific staining, cells were incubated with rabbit anti-MuHV-4 immune serum (1 h, 4°C), washed x2 in PBS, incubated with fluorescein-conjugated pig anti-rabbit pAb (Dako Cytomation) (1 h, 4°C), washed x2 in PBS and analysed on a FACS Calibur using Cellquest software (BD Biosciences).

Techniques: Infection, Staining, Expressing, Clone Assay

Journal: PLoS ONE

Article Title: An In Vitro System for Studying Murid Herpesvirus-4 Latency and Reactivation

doi: 10.1371/journal.pone.0011080

Figure Lengend Snippet: a . A latently infected TET-ON/OFF cell clone (c1.3) was analyzed for genome copy number by Southern blot (100 ng DNA) for the viral 1.2 kb Pst I terminal repeat fragment. For comparison we titrated purified MuHV-4 BAC DNA-estimated viral genome copy numbers are shown. 100 ng DNA is equivalent to approximately 10 4 cells. Therefore by this method c1.3 contained 1–3 viral genomes per cell. b . The viral genome copy number of 2 latently infected TET-ON/OFF cell clones (c1.3, c1.6) was determined by real-time PCR of 10 ng DNA. Values were normalized by the cellular APRT copy number of each sample to give the number of viral genomes per 2 copies of host DNA, i.e. per cell. Each point shows mean ± SD of triplicate samples. No change in copy number was observed over 3 weeks of continuous culture. UI = uninfected TET-ON/OFF cells. c . The viral genome copy numbers per 2 copies of cellular APRT were determined for c1.3 and c1.6 following doxycycline treatment (1 µg/ml). Each point shows mean ± SD of triplicate samples. UI = uninfected cells. d . c1.3 cells (TET-ON/OFF-TRE-50) were treated with doxycycline for the time indicated (0 h = no doxycycline), then lysed in situ . Circular and linear viral genomes were separated by electrophoresis, and Southern blots probed for the viral 1.2 kb terminal repeat. Controls included virion DNA (linear), BAC-cloned viral DNA (circular, although some has sheared to become linear) and TET-ON/OFF cells either uninfected (nil) or exposed overnight to wild-type MuHV-4 (+MuHV-4, mostly linear). The inset shows a longer exposure to reveal low copy number circular viral genomes.

Article Snippet: For specific staining, cells were incubated with rabbit anti-MuHV-4 immune serum (1 h, 4°C), washed x2 in PBS, incubated with fluorescein-conjugated pig anti-rabbit pAb (Dako Cytomation) (1 h, 4°C), washed x2 in PBS and analysed on a FACS Calibur using Cellquest software (BD Biosciences).

Techniques: Infection, Southern Blot, Purification, Clone Assay, Real-time Polymerase Chain Reaction, In Situ, Electrophoresis, Low Copy Number

Journal: PLoS ONE

Article Title: An In Vitro System for Studying Murid Herpesvirus-4 Latency and Reactivation

doi: 10.1371/journal.pone.0011080

Figure Lengend Snippet: a . Latently infected TET-ON/OFF cells were analyzed for MuHV-4 transcripts by RT-PCR before and after treatment with doxycycline (filled symbols). As a control, uninfected TET-ON/OFF cells were exposed to wild-type MuHV-4 (open symbols). Each point shows the mean ± SD copy number of triplicate RT-PCR reactions, adjusted to give the number of viral transcripts per 10 3 beta-actin transcripts in the same sample. Reverse transcriptase-negative controls always gave viral mRNA copy numbers that were undetectable or <1% of reverse transcriptase-positive samples. b . RNA (5 µg) was extracted from c1.3 cells either before induction (0 h) or after induction with doxycycline (1 µg/ml). UI = uninfected TET-ON/OFF cells. Replicate blots were probed for ORF50, M7 (a MuHV-4 late gene) or beta-actin.

Article Snippet: For specific staining, cells were incubated with rabbit anti-MuHV-4 immune serum (1 h, 4°C), washed x2 in PBS, incubated with fluorescein-conjugated pig anti-rabbit pAb (Dako Cytomation) (1 h, 4°C), washed x2 in PBS and analysed on a FACS Calibur using Cellquest software (BD Biosciences).

Techniques: Infection, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: An In Vitro System for Studying Murid Herpesvirus-4 Latency and Reactivation

doi: 10.1371/journal.pone.0011080

Figure Lengend Snippet: a . TET-ON/OFF cells latently infected with K3 + or K3 − TRE-50 MuHV-4 were treated with doxycyline (1 µg/ml), and virus titers determined by plaque assay 30 or 54 h later. Each point shows a separate clone. Infectious virus was undetectable without induction. b . Uninfected TET-ON/OFF cells were incubated with different doses of p56 peptide (2 h, 37°C), then washed x2 in DMEM and used to stimulate the MuHV-4-specific T cell hybridoma 49100.2 (16 h, 37°C). Beta-galactosidase production by the hybridoma cells was assayed by adding CPRG to cell lysates and reading the absorbance at 595 nm. Bars show mean ± SD of 3 replicate cultures. c . TET-ON/OFF clones either uninfected or latently infected with K3 + or K3 − TRE-50 MuHV-4 were incubated with doxycycline + phosphonoacetic acid (100 µg/ml) for the time incubated, then for a further 16 h with 49100.2 cells before assaying beta-galactosidase production as in b . Each bar shows mean ± SD of 3 replicate cultures for 1 clone. Hybridoma stimulation was significantly greater by reactivation K3 − MuHV-4 than by K3 + at all time points after induction (p<0.0001 by Student's 2-tailed t test). d . TET/ON-OFF cell clones carrying latent K3 + or K3 − TRE-50 MuHV-4 were induced with doxycycline without phosphonoacetic acid for the times indicated. MuHV-4-specific 49100.2 T cells were then added to each culture. Replicate cultures were assayed 16 h later for viral antigen recognition by beta-galactosidase assay, or for virus titer by plaque assay. Bars show mean ± SD of triplicate cultures for separate clones. K3 − clones were both recognized significantly better by 49100.2 cells and significantly inhibited in virus production compared to K3 + clones (p<0.01 by Student's 2-tailed t test).

Article Snippet: For specific staining, cells were incubated with rabbit anti-MuHV-4 immune serum (1 h, 4°C), washed x2 in PBS, incubated with fluorescein-conjugated pig anti-rabbit pAb (Dako Cytomation) (1 h, 4°C), washed x2 in PBS and analysed on a FACS Calibur using Cellquest software (BD Biosciences).

Techniques: Infection, Plaque Assay, Incubation, Clone Assay, β-Gal Assay

Journal: PLoS ONE

Article Title: The Leishmania ARL-1 and Golgi Traffic

doi: 10.1371/journal.pone.0001620

Figure Lengend Snippet: Consensus motives for the GDP-GTP binding site are boxed in black. Mutated and amino acids of interest are boxed in grey. The sequence of the C-terminal peptide synthesised for the production of the rabbit immune serum is underlined. Databanks accession numbers: Ld ARL-1, Genbank AF187855 and AAF29899; La ARL-1, GenBank XXX; Lm ARL-1, GeneDB LmjF17.0070 ( http://www.genedb.org/ ) ; Tb ARL-1, Genbank AAX70381, GeneDB Tb927.7.6230, previously Tb07.2F2.550; Hs ARL-1, Swissprot P40616; ScARL-1: Genbank S46035; Hs ARF-1: Swissprot P32889; Hs ARF-6: Genbank NP_001654.

Article Snippet: An anti- Ld ARL-1 C-terminus rabbit immune serum was prepared as described for Ld ARL-3A using an Ld ARL-1 C-terminal synthetic peptide (NH 2 -MDWLVERLREQGIGA-COOH, Genosphere Biotech) and a «Fauve de Bourgogne» rabbit (Centre d'Elevage Janvier).

Techniques: Binding Assay, Sequencing

Journal: PLoS ONE

Article Title: The Leishmania ARL-1 and Golgi Traffic

doi: 10.1371/journal.pone.0001620

Figure Lengend Snippet: Extracts of 3.10 6 exponentially growing L. amazonensis promastigotes (about 3 µg of proteins) were submitted to western blot analysis using the rabbit anti- Ld ARL-1 C-terminus immune serum (1∶5000 dilution); color was developed with an anti-rabbit IgG peroxidase conjugate (1∶10000 dilution) and an ECL revelation kit (Amersham). Internal standard: 37 kDa LACK antigen. Left, negative control without anti-LdARL-1 antiserum. Additional bands are revealed when the GFP-fused proteins are expressed, probably due to partial degradation when the cells were lysed, in spite of the presence of antiproteases (see ).

Article Snippet: An anti- Ld ARL-1 C-terminus rabbit immune serum was prepared as described for Ld ARL-3A using an Ld ARL-1 C-terminal synthetic peptide (NH 2 -MDWLVERLREQGIGA-COOH, Genosphere Biotech) and a «Fauve de Bourgogne» rabbit (Centre d'Elevage Janvier).

Techniques: Western Blot, Negative Control

Journal: PLoS ONE

Article Title: The Leishmania ARL-1 and Golgi Traffic

doi: 10.1371/journal.pone.0001620

Figure Lengend Snippet: A. IF on fixed cells: A1, phase contrast; A2, co-labelling with DAPI (blue) and FITC-anti rabbit-IgG plus anti- Ld ARL-1 rabbit immune serum (green). B. IF on fixed cells co-transformed with pNUS- Ld ARL-1-GFPcH (green) and pNUS-mREDnD- Ld LPG-2 (red) plus DAPI staining (blue). C. IF on fixed cells co-transformed with pNUS- Ld ARL-1-GFPcH (C1, green) plus pNUS- Ld ARF-1-mRedcD (C2, red), overlay (C3). D. EM of cells transformed with pNUS- Ld ARL-1-GFP and incubated with anti- Ld ARL-1 immune serum. C1 and C2 are sections of two different cells. Black dots (10 nm gold particles) show the Ld ARL-1 immunoreactive spots. N: nucleus; G: Golgi apparatus; K: kinetoplast; F: flagellum; FP: flagellar pocket.

Article Snippet: An anti- Ld ARL-1 C-terminus rabbit immune serum was prepared as described for Ld ARL-3A using an Ld ARL-1 C-terminal synthetic peptide (NH 2 -MDWLVERLREQGIGA-COOH, Genosphere Biotech) and a «Fauve de Bourgogne» rabbit (Centre d'Elevage Janvier).

Techniques: Transformation Assay, Staining, Incubation

Journal: PLoS ONE

Article Title: The Leishmania ARL-1 and Golgi Traffic

doi: 10.1371/journal.pone.0001620

Figure Lengend Snippet: Fixed L. amazonensis promastigotes transformed with: A, pNUS- Ld ARL-1-GFPcH (green); B, pNUS- Ld ARL-1/G2A-GFPcH (green).

Article Snippet: An anti- Ld ARL-1 C-terminus rabbit immune serum was prepared as described for Ld ARL-3A using an Ld ARL-1 C-terminal synthetic peptide (NH 2 -MDWLVERLREQGIGA-COOH, Genosphere Biotech) and a «Fauve de Bourgogne» rabbit (Centre d'Elevage Janvier).

Techniques: Transformation Assay

Journal: PLoS ONE

Article Title: The Leishmania ARL-1 and Golgi Traffic

doi: 10.1371/journal.pone.0001620

Figure Lengend Snippet: Fixed L. amazonensis promastigotes expressing in red Ld ARF-1-mRed (5B, 5E, 5H) and in green Ld ARL-1/Q74L-GFP (GTP) (5A), Ld ARL-1/T51N-GFP (GDP) (5D), Ld ARL-1/T34N-GFP (empty) (5H); overlays are in 5C, 5F and 5I. fp, flagellar pocket.

Article Snippet: An anti- Ld ARL-1 C-terminus rabbit immune serum was prepared as described for Ld ARL-3A using an Ld ARL-1 C-terminal synthetic peptide (NH 2 -MDWLVERLREQGIGA-COOH, Genosphere Biotech) and a «Fauve de Bourgogne» rabbit (Centre d'Elevage Janvier).

Techniques: Expressing

Journal: PLoS ONE

Article Title: The Leishmania ARL-1 and Golgi Traffic

doi: 10.1371/journal.pone.0001620

Figure Lengend Snippet: Fixed L. amazonensis promastigotes with plasma membrane labelled in red with Concanavalin A-Texas Red. A, cell expressing in green Ld ARL-1-GFP. B-C, cell expressing in green Ld ARL-1/T51N-GFP (GDP); D-F, cell expressing in green Ld ARL-1/T34N-GFP (empty). The yellow signal is an overlay of green and red signals.

Article Snippet: An anti- Ld ARL-1 C-terminus rabbit immune serum was prepared as described for Ld ARL-3A using an Ld ARL-1 C-terminal synthetic peptide (NH 2 -MDWLVERLREQGIGA-COOH, Genosphere Biotech) and a «Fauve de Bourgogne» rabbit (Centre d'Elevage Janvier).

Techniques: Expressing

Journal: PLoS ONE

Article Title: The Leishmania ARL-1 and Golgi Traffic

doi: 10.1371/journal.pone.0001620

Figure Lengend Snippet: L. amazonensis promastigotes expressing Ld ARL-1-GFP (A–D, green) or Ld ARL-1/T34N-GFP (empty) (E–H, green) were incubated at 24°C for 15 min with 2 µg/ml FM4-64FX (red), washed and further incubated for 0 min (A, E), 15 min (B, F), 60 min (C, G) and 120 min (D, H) before fixation; fp, flagellar pocket; MVT, Lysosome/Multivesicular Tubule. Colocalization of red and green results in a yellow signal.

Article Snippet: An anti- Ld ARL-1 C-terminus rabbit immune serum was prepared as described for Ld ARL-3A using an Ld ARL-1 C-terminal synthetic peptide (NH 2 -MDWLVERLREQGIGA-COOH, Genosphere Biotech) and a «Fauve de Bourgogne» rabbit (Centre d'Elevage Janvier).

Techniques: Expressing, Incubation

Journal: PLoS ONE

Article Title: The Leishmania ARL-1 and Golgi Traffic

doi: 10.1371/journal.pone.0001620

Figure Lengend Snippet: BA125 L. amazonensis promastigotes expressing in red the mRed-DPMS and in green Ld ARL-1-GFP (A), Ld ARL-1/G2A-GFP (unMyr) (B), Ld ARL-1/Q74L-GFP (GTP) (C), Ld ARL-1/T51N-GFP (GDP) (D) or Ld ARL-1/T34N-GFP (empty) (E).

Article Snippet: An anti- Ld ARL-1 C-terminus rabbit immune serum was prepared as described for Ld ARL-3A using an Ld ARL-1 C-terminal synthetic peptide (NH 2 -MDWLVERLREQGIGA-COOH, Genosphere Biotech) and a «Fauve de Bourgogne» rabbit (Centre d'Elevage Janvier).

Techniques: Expressing

Journal: PLoS ONE

Article Title: The Leishmania ARL-1 and Golgi Traffic

doi: 10.1371/journal.pone.0001620

Figure Lengend Snippet: A. Fixed L. amazonensis promastigotes expressing in green Ld ARL-1-GFP (1), Ld ARL-1/Q74L-GFP (GTP) (2), Ld ARL-1/T51N-GFP (GDP) (3), Ld ARL-1/G2A-GFP (unMyr) (4) or Ld ARL-1/T34N-GFP (empty) (5) and stained in red with the rabbit anti- Tb VP1 (acidocalcisomes marker) immune serum plus anti-rabbit IgG-Texas-Red conjugate. B. Live L. amazonensis promastigotes expressing in green Ld ARL-1-GFP (1-2), Ld ARL-1/T34N-GFP (empty) (3-4) or Ld ARL-1/T51N-GFP (GDP) (5-6) and stained with DAPI: in Blue, nucleus and kinetoplast, in yellow: polyphosphates of acidocalcisomes .

Article Snippet: An anti- Ld ARL-1 C-terminus rabbit immune serum was prepared as described for Ld ARL-3A using an Ld ARL-1 C-terminal synthetic peptide (NH 2 -MDWLVERLREQGIGA-COOH, Genosphere Biotech) and a «Fauve de Bourgogne» rabbit (Centre d'Elevage Janvier).

Techniques: Expressing, Staining, Marker

Journal: PLoS ONE

Article Title: The Leishmania ARL-1 and Golgi Traffic

doi: 10.1371/journal.pone.0001620

Figure Lengend Snippet: Overlay of fixed L. amazonensis promastigotes expressing in red mRed- Ld pGRIP-2 and in green Ld ARL-1-GFP (A), Ld ARL-1/Q74L-GFP (GTP) (B), Ld ARL-1/G2A-GFP (unMyr) (C), Ld ARL-1/T51N-GFP (GDP) (D) and Ld ARL-1/T34N-GFP (empty) (E). Overlay of red and green is yellow.

Article Snippet: An anti- Ld ARL-1 C-terminus rabbit immune serum was prepared as described for Ld ARL-3A using an Ld ARL-1 C-terminal synthetic peptide (NH 2 -MDWLVERLREQGIGA-COOH, Genosphere Biotech) and a «Fauve de Bourgogne» rabbit (Centre d'Elevage Janvier).

Techniques: Expressing