Journal:

Article Title: Poly(ADP-Ribose) Polymerase 1 Binds to Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Terminal Repeat Sequence and Modulates KSHV Replication in Latency

doi: 10.1128/JVI.78.18.9936-9946.2004

Figure Lengend Snippet: Western blot analysis of the TR(+)Sepharose bound and unbound fractions of BC3 and BJAB cell extracts. (A) Comparison of factors bound to the TR(+)Sepharose column. An aliquot of cell extract (CE; 0.1%), pass-through (Th; 0.01%), and KCl-eluted fractions of BC3 and BJAB cells were separated by SDS-4 to 20% PAGE, blotted on a polyvinylidene difluoride membrane, and analyzed with the commercially available antibodies shown to the left of the panel (α, anti). Typically detected bands are indicated by a bracket for LANA and arrows for ORC2, CDC6, Mcm7, and MSH6. (B) Comparison of bound and unbound fractions to TR(+)Sepharose and K-oriLyt CS Sepharose. K-oriLyt CS was also tested for TPA-uninduced or -induced materials. Binding of PARP1 and MSH6 to TR and K-oriLyt CS was examined. +, present; −, absent.

Article Snippet: Lanes 1, 4, 7, and 10, probe only; lanes 2, 5, 8, and 11, probes were incubated with 100 ng of human PARP1 (Trevigen); lanes 3, 6, 9, and 12, each probe was incubated with 100 ng of human PARP1 and a 100-fold excess of each nonlabeled probe.

Techniques: Western Blot, Comparison, Membrane, Binding Assay

Journal:

Article Title: Poly(ADP-Ribose) Polymerase 1 Binds to Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Terminal Repeat Sequence and Modulates KSHV Replication in Latency

doi: 10.1128/JVI.78.18.9936-9946.2004

Figure Lengend Snippet: PARP1 is colocalized with LANA. Anti-PARP1 (Alexa 546, red), anti-LANA (Alexa 488, green), and DAPI (blue) were used to stain nuclei, and merged images are also shown for each case. All of the data were obtained with a Zeiss 510 laser confocal fluorescent microscope. BC3 cells (A), BJAB cells (B), and 293 cells (C) expressing LANA and containing multiple copies of BAC TR-2, a TR-containing bacterial artificial chromosome construct.

Article Snippet: Lanes 1, 4, 7, and 10, probe only; lanes 2, 5, 8, and 11, probes were incubated with 100 ng of human PARP1 (Trevigen); lanes 3, 6, 9, and 12, each probe was incubated with 100 ng of human PARP1 and a 100-fold excess of each nonlabeled probe.

Techniques: Staining, Microscopy, Expressing, Construct

Journal:

Article Title: Poly(ADP-Ribose) Polymerase 1 Binds to Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Terminal Repeat Sequence and Modulates KSHV Replication in Latency

doi: 10.1128/JVI.78.18.9936-9946.2004

Figure Lengend Snippet: PARP1-binding assay. (A) Schematic representation of the TR fragments used for PARP1-binding experiments with ELISA and EMSA. Constructs 1, 2, 2-1, 2-1-1, and 2-1-2 were used in ELISAs, and constructs 2-2 to 2-5 were used in EMSAs. The position of the LANA-binding sequence (LBS) and the suspected PARP1-binding sequences (5′-TCGNT-3′) reported by Nirodi et al. (41) are also shown. (B) PARP1 binding by ELISA. The bound biotinylated fragment was detected colorimetrically by using a streptavidin-biotin-AP complex and pNpp as a substrate. The optical density at 405 nm was measured, and the arbitrary scale of binding intensity for each fragment is shown, where the intensity for the full-length TR was set at 1. (C) EMSA. Each fragment was fill-in labeled with Klenow fragment and [α-32P]dCTP. Lanes 1, 4, 7, and 10, probe only; lanes 2, 5, 8, and 11, probes were incubated with 100 ng of human PARP1 (Trevigen); lanes 3, 6, 9, and 12, each probe was incubated with 100 ng of human PARP1 and a 100-fold excess of each nonlabeled probe. Specifically shifted bands are indicated by arrowheads. (D) EMSA with specific antibodies against PARP1. In this analysis, constructs 2-2 and 2-3 were analyzed. Lanes 1 and 6, probes only; lanes 2 and 7, probes were incubated with 100 ng of human PARP1; lanes 3 and 8, probes were incubated with rabbit polyclonal anti-PARP1 (αPARP1) antibodies (2 μg) and 100 ng of human PARP1; lanes 4 and 9, probes were incubated with normal rabbit IgG (2 μg) and 100 ng of human PARP1; lanes 5 and 10, probes were incubated with 100 ng of human PARP1 and a 100-fold excess of nonlabeled probes. The band that specifically shifted and disappeared with the addition of antibody is indicated by an arrowhead. Asterisks denote nonspecific binding. +, present; −, absent.

Article Snippet: Lanes 1, 4, 7, and 10, probe only; lanes 2, 5, 8, and 11, probes were incubated with 100 ng of human PARP1 (Trevigen); lanes 3, 6, 9, and 12, each probe was incubated with 100 ng of human PARP1 and a 100-fold excess of each nonlabeled probe.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Construct, Sequencing, Labeling, Incubation

Journal:

Article Title: Poly(ADP-Ribose) Polymerase 1 Binds to Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Terminal Repeat Sequence and Modulates KSHV Replication in Latency

doi: 10.1128/JVI.78.18.9936-9946.2004

Figure Lengend Snippet: Poly(ADP-ribosyl)ation assay of LANA. (A) Immunoprecipitation followed by Western blot analysis. Whole-cell extracts (WCE) from BC3 cells were immunoprecipitated (IP) with the designated antibodies (2 μg of each) and immunoblotted (IB) with a rat monoclonal anti-LANA antibody (left) or rabbit polyclonal anti-PAR antibodies (right). Multiple forms of LANA are indicated. The arrowhead shows poly(ADP-ribosyl)ated LANA detected by the PAR antibodies. (B) Biotinylated NAD incorporation assay. LANA was immunoprecipitated with either a rat monoclonal anti-LANA (αLANA) antibody (2 μg) or normal rat IgG (2 μg). The immunoprecipitates were incubated with biotinylated NAD and 100 ng of purified human PARP1 (Trevigen) as described in Materials and Methods. The samples were then fractionated by SDS-PAGE and blotted with either a rat monoclonal anti-LANA antibody (2 μg) (left) or streptavidin-HRP (right). The immunoprecipitated LANA (left) and NAD-incorporated LANA and PARP1 are indicated by arrowheads. +, present; −, absent.

Article Snippet: Lanes 1, 4, 7, and 10, probe only; lanes 2, 5, 8, and 11, probes were incubated with 100 ng of human PARP1 (Trevigen); lanes 3, 6, 9, and 12, each probe was incubated with 100 ng of human PARP1 and a 100-fold excess of each nonlabeled probe.

Techniques: Immunoprecipitation, Western Blot, Incubation, Purification, SDS Page

Journal:

Article Title: Poly(ADP-Ribose) Polymerase 1 Binds to Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Terminal Repeat Sequence and Modulates KSHV Replication in Latency

doi: 10.1128/JVI.78.18.9936-9946.2004

Figure Lengend Snippet: Viral genome copy number and cell viability in BC3 cells treated with drugs affecting PARP activity. BC3 cells were untreated (NT) or treated with NA or HU as described in Materials and Methods and tested for viral genome copy number by real-time PCR (A) and for cell viability by the trypan blue method (B). (A) Viral genome copy number in drug-treated cells. The viral genome copy number was estimated by amplifying the K-oriLyt CS region and normalized to the β-globin gene content by real-time PCR on the designated day. The number of copies is shown on a logarithmic scale as viral copy number per picogram of DNA. The value above the bar shows a mean value for each case. (B) Cell viability of dru-treated cells. The viable cells were counted on the designated day as described. (C) HU treatment leads to the increase in poly(ADP-ribosyl)ation of LANA. BC3 cells were treated with NT (nontreatment), TPA (25 ng/ml; Sigma), HU (50 μM), or NA (10 mM), and 3 days after treatment, cells were harvested and solubilized with RIPA buffer and the soluble fraction was immunoprecipitated (IP) with either a preimmune rat IgG or a rat monoclonal anti-LANA antibody. The immunoprecipitated fraction was fractionated by SDS-PAGE and immunoblotted (IB) with either rabbit polyclonal anti-PAR (αPAR) antibodies or a rat monoclonal anti-LANA (αLANA) antibody. (D) No lytic gene is expressed by drug treatment. BC3 cells were fixed with 4% paraformaldehyde-PBS 1 day after treatment with NT (nontreatment), TPA (25 ng/ml; Sigma), HU (50 μM), or NA (10 mM). Then the cells were incubated with a mouse monoclonal anti-RTA antibody, followed by goat anti-mouse IgG conjugated with Alexa 488 (Molecular Probes-Invitrogen) (green signal). The signal was detected with a confocal fluorescence microscope (Zeiss 510) (original magnification, ×600). The nucleus was counterstained with DAPI.

Article Snippet: Lanes 1, 4, 7, and 10, probe only; lanes 2, 5, 8, and 11, probes were incubated with 100 ng of human PARP1 (Trevigen); lanes 3, 6, 9, and 12, each probe was incubated with 100 ng of human PARP1 and a 100-fold excess of each nonlabeled probe.

Techniques: Activity Assay, Real-time Polymerase Chain Reaction, Immunoprecipitation, SDS Page, Incubation, Fluorescence, Microscopy