baculovirus expression constructs  (GenScript corporation)

Journal: Nucleic Acids Research

Article Title: Analyzing structure–function relationships of artificial and cancer-associated PARP1 variants by reconstituting TALEN-generated HeLa PARP1 knock-out cells

doi: 10.1093/nar/gkw859

Figure Lengend Snippet: TALEN-mediated gene targeting of PARP1 in HeLa cells. ( A ) Single-cell immuno-epifluorescence analysis of PARP1 expression in HeLa WT and in two independently generated PARP1 knock-out (KO) clones (KO1 and KO2). ( B ) Western blot analysis of PARP1 expression in HeLa WT and PARP1 KO clones. PCNA served as a loading control. ( C ) Single cell immuno-epifluorescence analysis of PAR formation in HeLa WT and PARP1 KO clones. WT cells showed a dose-dependent increase in cellular PAR levels upon H 2 O 2 treatment (for 5 min), while PAR levels in PARP1 KO cells remained close to background signal intensities. Representative epifluorescent microscopic images (left panel), quantitation of image data (right panel). Means ± SEM, at least 70 cells per data point were analyzed. Statistical analysis was performed via two-way ANOVA testing and Sidak's post-test. ( D) Intracellular NAD + levels in WT and PARP1 KO cells ± H 2 O 2 treatment for 7 min as measured by an enzymatic NAD + cycling assay. Means ± SEM of n = 3 independent experiments. Statistical analysis was performed using two-way ANOVA testing and Sidak's post-test. ( E ) Quantitation of basal and H 2 O 2 -induced PAR levels in WT and PARP1 KO cells via isotope dilution mass spectrometry (LC–MS/MS) using a previously published method . To induce PAR-formation, cells were treated with H 2 O 2 as indicated. If indicated, cells were pretreated with 10 μM ABT888 for 45 min. Insert: Basal PAR levels in untreated WT and PARP1 KO cells. Means ± SEM of n = 3 independent experiments. Statistical analysis was performed using one-way ANOVA testing and Sidak's post-test within one group of cells (i.e. WT, KO1, KO2). ( F ) LC–MS/MS analysis of PAR levels ± camptothecin (CPT) treatment for 30 min. Means of n = 2 independent experiments. R-Ado indicates ribosyl-adenosine.

Article Snippet: Baculovirus expression constructs of PARP1 were generated according to manufacturer's instructions (BD).

Techniques: Expressing, Generated, Knock-Out, Clone Assay, Western Blot, Control, Quantitation Assay, Isotope Dilution, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

Journal: Nucleic Acids Research

Article Title: Analyzing structure–function relationships of artificial and cancer-associated PARP1 variants by reconstituting TALEN-generated HeLa PARP1 knock-out cells

doi: 10.1093/nar/gkw859

Figure Lengend Snippet: Functional consequences of PARP1 deletion in HeLa cells. ( A ) Cell proliferation of HeLa WT and PARP1 KO cells as analyzed by Alamar Blue assay for 3 days. Means ± SEM of n = 3 independent experiments. Statistical analysis was performed using two-way ANOVA testing and Sidak's post-test. ( B ) Cell cycle analysis of untreated WT and PARP1 KO cells via PI staining and subsequent flow cytometric analysis. Means ± SEM of three independent experiments. ( C ) Clonogenic survival analysis. HeLa WT and PARP1 KO cells were treated with H 2 O 2 as indicated for 5 min and then plated and cultivated for 2 weeks prior to colony counting. Means ± SEM of n = 3 independent experiments. Statistical analysis using two-way ANOVA testing and Sidak's post-test. ( D ) Cytotoxicity analysis via annexin V/PI staining and subsequent flow cytometric analysis of HeLa WT and PARP1 KO cells treated ± CPT in concentrations as indicated for 2 days. Viable cells refer to annexin V/PI-double negative cells (top); early apoptotic cells to annexin V-positive (middle), PI-negative cells; and necrotic and late-apoptotic cells to annexin V/PI-double positive cells (bottom). Ratios compared to total cell numbers. Means ± SEM of n ≥ 4 independent experiments. Statistical analysis was performed using two-way ANOVA testing and Sidak's post-test. ( E ) Cell cycle analysis via PI staining and flow cytometric analysis 2 days after treatment of cells ± CPT in concentrations as indicated. Means ± SEM of n ≥ 4 independent experiments except for data of PARP1 KO2 cells; n = 1. Statistical analysis was performed using two-way ANOVA testing and Sidak's post-test.

Article Snippet: Baculovirus expression constructs of PARP1 were generated according to manufacturer's instructions (BD).

Techniques: Functional Assay, Alamar Blue Assay, Cell Cycle Assay, Staining

Journal: Nucleic Acids Research

Article Title: Analyzing structure–function relationships of artificial and cancer-associated PARP1 variants by reconstituting TALEN-generated HeLa PARP1 knock-out cells

doi: 10.1093/nar/gkw859

Figure Lengend Snippet: Overview of PARP1 variants included in this study. ( A ) PARP1 structure and localization of amino acid exchanges of PARP1 variants as used in this study. The 3D structure is in complex with a double-stranded DNA molecule [PDB code 4DQY ], without ZnF2 and WGR domains. ( B ) Biochemical parameters derived from rec. enzymes of the different PARP1 variants used in this study. Values were determined in the present study ( cf . Figure ) or taken from the literature as indicated. PAR indicates poly(ADP-ribose); MAR, mono(ADP-ribose); and OAR, oligo(ADP-ribose). ( C ) Western blot analysis of PARP1 protein levels in HeLa PARP1 KO cells reconstituted with different PARP1-eGFP variants 2 days after transfection. Left: representative Western blot out of 4. Right: densitometric analysis of western blot signal intensities after normalization to transfection efficiencies. Means ± SEM of n = 4 independent experiments. Statistical analysis was performed using 1-sample t -test comparing the expression of the different PARP1-variants after transfection to endogenous PARP1\WT-levels in HeLa cells.

Article Snippet: Baculovirus expression constructs of PARP1 were generated according to manufacturer's instructions (BD).

Techniques: Derivative Assay, Western Blot, Transfection, Expressing

Journal: Nucleic Acids Research

Article Title: Analyzing structure–function relationships of artificial and cancer-associated PARP1 variants by reconstituting TALEN-generated HeLa PARP1 knock-out cells

doi: 10.1093/nar/gkw859

Figure Lengend Snippet: Cellular biochemistry of artificial PARP1 mutants. HeLa PARP1 KO cells were transfected with eGFP-coupled constructs of PARP1, PARP1\L713F and PARP1\E988K. Analyses were performed 2 days after transfection. ( A ) Representative images from single cell immuno-epifluorescence analysis of PARP1-eGFP and PAR after treatment of PARP1-reconstituted cells ± H 2 O 2 as indicated for 5 min. Scale bars indicate 30 μm. ( B ) Densitometric analysis of imaging data as shown in (A). More than 100 cells were analyzed per experiment and condition in a semi-automated manner using a KNIME workflow. Means ± SEM of n = 4 independent experiments. Statistical analysis using matched two-way ANOVA testing and Sidak's post-test. ( C ) Time-course analysis of PAR levels in PARP1-reconstituted cells after treatment of cells with 250 μM H 2 O 2 . Means ± SEM of n = 4 independent experiments, >100 cells were analyzed per experiment and condition. Statistical analysis was performed using matched two-way ANOVA testing and Sidak's post-test. ( D ) LC–MS/MS analyses of PAR levels in PARP1 KO1 cells and cells reconstituted with PARP1\WT and PARP1\L713F. Two days after transfection, cells were treated as indicated for 7 min. Levels were normalized to transfection efficiencies. Means ± SEM of n = 3 independent experiments. Statistical analysis was performed by one-way ANOVA testing and Tukey's post-test. ( E ) Western blot analysis of HeLa cell extracts of KO1 and PARP1-reconstituted cells, as indicated. 2 days after transfection, cells were treated with 500 μM H 2 O 2 for 7 min. PARylated proteins were detected via the 10H antibody. Red arrows indicate the expected molecular weight of auto-PARylated PARP1. ( F ) NAD + levels in PARP1-reconstituted cells upon treatment ± H 2 O 2 for 7 min as measured by an enzymatic NAD + cycling assay. Means ± SEM of n = 3 independent experiments, except for ABT888-treated samples, n = 2. Statistical analysis was performed using two-way ANOVA testing and Sidak's post-test.

Article Snippet: Baculovirus expression constructs of PARP1 were generated according to manufacturer's instructions (BD).

Techniques: Transfection, Construct, Imaging, Liquid Chromatography with Mass Spectroscopy, Western Blot, Molecular Weight

Journal: Nucleic Acids Research

Article Title: Analyzing structure–function relationships of artificial and cancer-associated PARP1 variants by reconstituting TALEN-generated HeLa PARP1 knock-out cells

doi: 10.1093/nar/gkw859

Figure Lengend Snippet: Recruitment and dissociation kinetics of PARP1-eGFP at sites of laser-induced DNA damage. ( A ) Representative imaging data. Scale bars indicate 10 μm. ( B ) Densitometric quantitation of signal intensities from imaging data as shown in (A). Means ± SEM of n = 3 independent experiments, >29 cells were analyzed per experiment and condition. Statistical analysis was performed using two-way ANOVA testing and Sidak's post-test.

Article Snippet: Baculovirus expression constructs of PARP1 were generated according to manufacturer's instructions (BD).

Techniques: Imaging, Quantitation Assay

Journal: Nucleic Acids Research

Article Title: Analyzing structure–function relationships of artificial and cancer-associated PARP1 variants by reconstituting TALEN-generated HeLa PARP1 knock-out cells

doi: 10.1093/nar/gkw859

Figure Lengend Snippet: Non-covalent PARP1–PAR interaction. ( A ) Analysis of PARP1–PAR interaction by PAR overlay assay using increasing amounts of recombinant PARP1 as indicated. After protein transfer, membranes were incubated with (left panel) or without (right panel) purified PAR (0.2 μM). PAR binding was detected using the 10H antibody after high-stringency washing to remove non-specifically bound PAR. ( B ) Immuno slot-blot PAR binding assay using increasing amounts of recombinant PARP1. Membranes were incubated with PAR (0.2 μM) and bound PAR was detected using the 10H antibody after high-stringency washing. ( C ) Analysis of PARP1–PAR interaction in solution using a modified PAR-EMSA. End-biotinylated PAR of defined chain length (30–35 mer, 0.5 pmol ADP-ribose) was incubated with recombinant PARP1. PARP1–PAR binding was assessed by native TBE gel electrophoreses and Western blotting. Three distinct complexes (1–3) were formed in a PARP1-dependent manner. ( D ) Upper panel. In silico search for putative PAR-binding sites within the PARP1 sequence using the search sequence displayed at the top of the panel. Two potential PAR binding motives (PBMs), i.e. PBM1 (1 mismatch) and PBM2 (two mismatches), were identified in Zn2 and Zn3, respectively. Lower panels . Localization of PBM1 and PBM2 within Zn2 and Zn3, respectively. Structures based on PDB codes 4AV1 and 4DQY . ( E ) PAR binding ability of peptides comprising aa sequences of PBM1/2 and peptides comprising aa exchanges potentially responsible of PBM-PAR interactions using a PepSpot analysis. ‘AA pos.’ indicates aa positions within full-length PARP1 sequence . A peptide sequence derived from a PBM in XRCC1 served as a positive control. ( F ) DNA-PARP1 EMSA using a known biotinylated double-stranded DNA oligonucleotide (200 fmol). Left . DNA-PARP1 interaction in the absence of PAR. Middle . Rec. PARP1 (100 nM) was incubated with increasing concentrations of PAR as indicated. Right . Densitometric evaluation of EMSAs. Means ± SEM of n = 3 independent experiments. Statistical analysis was performed using 1-way ANOVA testing and Sidak's post-test.

Article Snippet: Baculovirus expression constructs of PARP1 were generated according to manufacturer's instructions (BD).

Techniques: Overlay Assay, Recombinant, Incubation, Purification, Binding Assay, Dot Blot, Modification, Western Blot, In Silico, Sequencing, Derivative Assay, Positive Control

Journal: Nucleic Acids Research

Article Title: Analyzing structure–function relationships of artificial and cancer-associated PARP1 variants by reconstituting TALEN-generated HeLa PARP1 knock-out cells

doi: 10.1093/nar/gkw859

Figure Lengend Snippet: PARP1\E988K affects cell cycle regulation and induces DNA damage signaling. ( A ) Cell cycle analysis by PI staining and subsequent flow cytometric analysis 3 days after transfection of HeLa PARP1 KO cells reconstituted with PARP1\WT, PARP1\E988K and PARP1\L713F. PARP1\E988K induces a G2 arrest, which can be rescued by treating cells with 10 μM ABT888. Means ± SEM of n = 6 independent experiments, except of ABT888-treated samples, n = 2. Statistical analysis was performed using two-way ANOVA testing and Sidak's post-test. ( B ) Analysis of DNA damage response markers, i.e. phospho-S15-p53, γH2A.X and p16 in WT, PARP1 KO and PARP1-reconstituted cells as indicated 2 days after transfection. Cells reconstituted with PARP1\E988K displayed increased phospho-S15-p53 and γH2A.X levels and cells reconstituted with PARP1\L713F showed slightly elevated γH2A.X levels. Immunochemical detection of PARP1, p53 and actin served as controls. Shown is a representative experiment out of three. ( C ) Western blot analysis of ph-p53 and γH2A.X levels in PARP1-reconstituted cells (±10-μM ABT888 treatment). ( D ) Western blot analysis of the replicative stress marker phospho-RPA2 (Ser4/8) in PARP1-reconstituted cells (±10-μM ABT888 treatment). Immunochemical detection of PARP1 and actin served as controls.

Article Snippet: Baculovirus expression constructs of PARP1 were generated according to manufacturer's instructions (BD).

Techniques: Cell Cycle Assay, Staining, Transfection, Western Blot, Marker

Journal: Nucleic Acids Research

Article Title: Analyzing structure–function relationships of artificial and cancer-associated PARP1 variants by reconstituting TALEN-generated HeLa PARP1 knock-out cells

doi: 10.1093/nar/gkw859

Figure Lengend Snippet: PARP1 mutants influence cell viability and cell cycle progression upon CPT treatment. ( A and B ) Analysis of viable, apoptotic and necrotic cells 3 days after transfection of HeLa PARP1 KO cells reconstituted with PARP1, PARP1\E988K, and PARP1\L713F by annexinV / PI staining and subsequent flow cytometric analysis. GFP cont indicates cells transfected with a plasmid carrying only GFP. Cells were treated ( A ) with CPT in concentrations as indicated 24 h after transfection or ( B ) with 10 μM ABT888 directly after transfection. Viable cells refer to annexin V/PI-double negative cells; (early) apoptotic cells to annexin V-positive, PI-negative cells; and necrotic and late-apoptotic cells to annexin V/PI-double positive cells (ratios compared to total cell numbers). Means ± SEM of n ≥ 3 independent experiments. Statistical analysis was performed using two-way ANOVA testing and Sidak's post-test. ( C ) Cell cycle analysis of PARP1-reconstituted cells as indicated 3 days after transfection and 2 days after CPT treatment via PI staining and subsequent flow cytometric analysis. Means ± SEM of n = 3 independent experiments. Statistical analysis was performed using two-way ANOVA testing and Sidak's post-test.

Article Snippet: Baculovirus expression constructs of PARP1 were generated according to manufacturer's instructions (BD).

Techniques: Transfection, Staining, Plasmid Preparation, Cell Cycle Assay

Journal: Nucleic Acids Research

Article Title: Analyzing structure–function relationships of artificial and cancer-associated PARP1 variants by reconstituting TALEN-generated HeLa PARP1 knock-out cells

doi: 10.1093/nar/gkw859

Figure Lengend Snippet: Biochemical and cellular characteristics of natural PARP1 variants. ( A ) An inherited PARP1 mutation identified by exome sequencing in a patient with pediatric colorectal cancer. The panel on the left shows a subset of the sequencing reads spanning the individual mutations (data based on hg19); the panel on the right shows the validation by Sanger sequencing in the child and the maternal samples to indicate the mode of inheritance. Position of the mutation is indicated by red arrows. ( B ) Biochemical characterization of natural PARP1 variants as used in this study. Rec. enzymes were expressed in the Sf 9/baculovirus system and purified via size exclusion and affinity chromatography. PARP1 activity was examined by incubating 5 nM PARP1 with increasing concentrations of NAD + as indicated in a reaction mixture as described in material and methods section. Afterward, 15% of reaction mixtures were slot-blotted on a nylon membrane (see Supplementary Figure S7) and PAR content was analyzed by immunochemical staining using the 10H antibody. Means of n = 3 independent experiments. A non-linear Michaelis-Menten model was used for curve fit. Statistical analysis using 2-way ANOVA testing. ( C ) Analysis of intracellular PARP1 activity in PARP1 KO cells reconstituted with PARP1 variants as indicated 2 days after transfection by immuno-epifluorescence microscopy as shown in Figure (for representative raw data refer to Supplementary Figure S7). Cells were treated with H 2 O 2 for 5 min in concentrations as indicated, and PAR levels of eGFP-positive cells were examined using the anti-PAR-specific mAB 10H. Means ± SEM of n = 5 independent experiments. Statistical analysis was performed using matched two-way ANOVA testing and Sidak's post-test. ( D ) Time-course analysis of PAR formation in PARP1-reconstituted cells after treatment of cells with 250 μM H 2 O 2 . Means ± SEM of n = 4 independent experiments (>100 cells per experiment). Statistical analysis using matched two-way ANOVA testing and Sidak's post-test. ( E ) NAD + levels in WT, PARP1 KO and PARP1-reconstituted cells ± H 2 O 2 treatment for 7 min as evaluated by an enzymatic NAD + cycling assay. Means ± SEM of n = 3 independent experiments. Statistical analysis was performed via 2-way ANOVA testing and Sidak's post-test. ( F ) Recruitment and dissociation kinetics of natural PARP1 variants at sites of laser-induced DNA damage. For representative raw data refer to Supplementary Figure S7. Means ± SEM. Evaluation from ≥35 cells from three independent experiments. Statistical analysis was performed using two-way ANOVA testing and Sidak's post-test.

Article Snippet: Baculovirus expression constructs of PARP1 were generated according to manufacturer's instructions (BD).

Techniques: Mutagenesis, Sequencing, Biomarker Discovery, Purification, Affinity Chromatography, Activity Assay, Membrane, Staining, Transfection, Epifluorescence Microscopy

Journal: Journal of Virology

Article Title: Characterization of an Amino-Terminal Dimerization Domain from Retroviral Restriction Factor Fv1

doi: 10.1128/jvi.00395-06

Figure Lengend Snippet: FIG. 1. Analysis of the expression, purification, and in vivo proteolysis of Fv1n in recombinant baculovirus-infected insect cells. (A) Coomassie brilliant blue-stained SDS gel of nickel-chelate affinity purification. M, molecular weight markers; Wh, whole-cell extract; S, soluble fraction; P, pellet fraction; W, wash; E1 to E3, fractions from imidazole elution. (B) Western blot showing purified Fv1n (A) and copurifying fragments (B to G). (C) Table of the N-terminal sequences determined by automated Edman degradation. Fragments and Fv1n are labeled as in panel B. Relative molecular weights (Mr) are in thousands. Ct, C-terminal.

Article Snippet: An Fv1n baculovirus expression construct was prepared using the BD BacPAK baculovirus expression system from Clontech.

Techniques: Expressing, In Vivo, Recombinant, Infection, Staining, SDS-Gel, Molecular Weight, Western Blot, Labeling

Journal: Journal of Virology

Article Title: Characterization of an Amino-Terminal Dimerization Domain from Retroviral Restriction Factor Fv1

doi: 10.1128/jvi.00395-06

Figure Lengend Snippet: FIG. 3. Fractionation of Fv1n and copurifying fragments by size-exclusion chromatography. (A) Chromatogram produced by fractionation of nickel-chelate affinity-purified Fv1n on a Superdex 200 (10/30) column. The column was calibrated with standards, and the expected molecular masses (kDa) based on this calibration together with the column void volume (Vo) are indicated. (B) Western blot analysis using anti-Fv1 antibodies of fractions eluted from the size-exclusion column. The lane number corresponds to the fraction number in panel A, Ld is the material loaded onto the column, and the lanes marked M contained molecular weight markers. (C) Elution profile of purified Fv1(20-200) applied to a Superdex 200 (10/30) column. (D) SDS-PAGE of the peak fractions from panel C, the gel is stained with Coomassie brilliant blue, and the molecular masses (kDa) of the size markers are indicated.

Article Snippet: An Fv1n baculovirus expression construct was prepared using the BD BacPAK baculovirus expression system from Clontech.

Techniques: Fractionation, Size-exclusion Chromatography, Produced, Western Blot, Molecular Weight, SDS Page, Staining