Journal: Cells

Article Title: PARP1, DIDO3, and DHX9 Proteins Mutually Interact in Mouse Fibroblasts, with Effects on DNA Replication Dynamics, Senescence, and Oncogenic Transformation

doi: 10.3390/cells13020159

Figure Lengend Snippet: Interaction of DIDO3 with PARP1 and DHX9. ( A ) Western blots of samples from transfected NIH-3T3 cells expressing GFP, as a negative control, or HA-tagged-full-length DIDO3, -DIDO2 or -DIDO3-CT, untreated (−) or treated with Olaparib (+). The cells were lysed (left) and immunoprecipitated with anti-HA antibody (right); the blots were probed for PARP1, DHX9, and HA-tagged proteins. Olaparib treatment increased the co-precipitation of both proteins only in the case of full-length DIDO3 (red box). ( B ) Western blots of WT and DIDO3 mutant (dE16) cell lysates and their precipitates, previously untreated (−) or treated with Olaparib (+). Precipitation was performed with magnetic beads covalently linked to Af1521 Macrodomain, which specifically binds to PARylated proteins. The blots were probed with anti-DHX9 antibody (upper), anti-PARP1 antibody (middle) and anti-PAR antibody (lower panel).

Article Snippet: Af1521 Macrodomain Magnetic Resin (Tulip Biolabs, Lansdale, PA, USA) was used to precipitate mono- and poly-ADP ribosylated proteins [ ], according to the manufacturer’s protocol.

Techniques: Western Blot, Transfection, Expressing, Negative Control, Immunoprecipitation, Mutagenesis, Magnetic Beads

Journal: Journal of Proteome Research

Article Title: A Study into the ADP-Ribosylome of IFN-γ-Stimulated THP-1 Human Macrophage-like Cells Identifies ARTD8/PARP14 and ARTD9/PARP9 ADP-Ribosylation

doi: 10.1021/acs.jproteome.8b00895

Figure Lengend Snippet: ADP-ribosylation increases during IFN-γ-induced pro-inflammatory activation of macrophages. (A) Two independent strategies, Af1521 and 10H antibody workflows, for ADP-ribosylation proteomics. (B) Antipan ADP-ribose Western blot analysis of IFN-γ-treated THP-1 cells over 24 h. (C) IFN-γ activation replicates: two sets (control or IFN-γ) of macrophage activation were used for the Af1521 workflow, and three sets were used for the 10H antibody workflow. Details about ADPr peptide data acquisition and analysis are highlighted using the second Af1521 replicate of IFN-γ-treated THP-1 cells.

Article Snippet: Cell lysate from three 10 cm-dishes (1.0 mg) was incubated with 150 μL of Af1521 macrodomain conjugated to Glutathione sepharose 4B (GE Healthcare, Chicago, IL, Cat# 17075601) for 90 min at 4 °C using a rotator.

Techniques: Activation Assay, Western Blot

Journal: Journal of Proteome Research

Article Title: A Study into the ADP-Ribosylome of IFN-γ-Stimulated THP-1 Human Macrophage-like Cells Identifies ARTD8/PARP14 and ARTD9/PARP9 ADP-Ribosylation

doi: 10.1021/acs.jproteome.8b00895

Figure Lengend Snippet: Gas phase segmentation (GPS) improves ADPr peptide detection. (A) A screenshot of the ADP-ribose product ion triggered EThcD and HCD data acquisition method on the Orbitrap Fusion Lumos. (B) Area under the curve (AUC) of the four ADPr fragment ions (136.06, 250.09, 348.07, and 428.04 m / z ) dissociated from an ARTD8/PARP14-MARylated STAT1 peptide using HCD. (C) Pilot Af1521 enrichment study to determine the optimal collision energy for ADP-ribose product ion screening and ADPr peptide identification. Only high confidence (HCD and EThcD combined peptides) were used in this plot (more details in Figure ). (D) A schematic showing the principle of GPS using multiple injections. (E) The extracted ion chromatograms of the ADPr fragment peak (348.07 m / z ) in each full scan and GPS injection. (F,G) Precursor ion m / z and retention time of triggered EThcD spectra in full scan and combined GPS scans.

Article Snippet: Cell lysate from three 10 cm-dishes (1.0 mg) was incubated with 150 μL of Af1521 macrodomain conjugated to Glutathione sepharose 4B (GE Healthcare, Chicago, IL, Cat# 17075601) for 90 min at 4 °C using a rotator.

Techniques: Injection

Journal: Journal of Proteome Research

Article Title: A Study into the ADP-Ribosylome of IFN-γ-Stimulated THP-1 Human Macrophage-like Cells Identifies ARTD8/PARP14 and ARTD9/PARP9 ADP-Ribosylation

doi: 10.1021/acs.jproteome.8b00895

Figure Lengend Snippet: Data processing of product ion triggered MS/MS spectra. (A) A schematic of SEQUEST-HT searches of triggered EThcD and HCD spectra using the second Af1521 replicate of IFN-γ-treated THP-1 cells. (B) Number of peptide-spectrum matches (PSMs) of assigned ADPr and unmodified peptides from the triggered spectra. (C–E) Distribution of isolation interference for product ion triggered or DDA PSMs. (F) Number of ADPr peptides with high confidence detected by either EThcD or HCD. (G) Venn diagrams comparing ADPr peptide identifications between EThcD and HCD for all ADPr peptides, and those with >95% ADPr acceptor site probability.

Article Snippet: Cell lysate from three 10 cm-dishes (1.0 mg) was incubated with 150 μL of Af1521 macrodomain conjugated to Glutathione sepharose 4B (GE Healthcare, Chicago, IL, Cat# 17075601) for 90 min at 4 °C using a rotator.

Techniques: Tandem Mass Spectroscopy, Isolation

Journal: Journal of Proteome Research

Article Title: A Study into the ADP-Ribosylome of IFN-γ-Stimulated THP-1 Human Macrophage-like Cells Identifies ARTD8/PARP14 and ARTD9/PARP9 ADP-Ribosylation

doi: 10.1021/acs.jproteome.8b00895

Figure Lengend Snippet: EThcD provides improved ADPr acceptor site localization in THP-1 protein substrates. (A) Distribution of confident ADPr acceptor amino acids (>95% site probability) of all identified ADPr peptides triggered by either EThcD or HCD spectra using the second Af1521 replicate of IFN-γ-treated THP-1 cells. (B) Distribution of confident ADPr acceptor amino acids of the 57 common ADPr peptides ( Figure G). (C) An alluvial diagram showing conserved or variant assignments between the two activation methods. (D,E) ARTD1/PARP1 ADPr annotated spectra. ADPr-associated peaks (black peaks) were manually annotated. *, ADPr site.

Article Snippet: Cell lysate from three 10 cm-dishes (1.0 mg) was incubated with 150 μL of Af1521 macrodomain conjugated to Glutathione sepharose 4B (GE Healthcare, Chicago, IL, Cat# 17075601) for 90 min at 4 °C using a rotator.

Techniques: Variant Assay, Activation Assay

Journal: Journal of Proteome Research

Article Title: A Study into the ADP-Ribosylome of IFN-γ-Stimulated THP-1 Human Macrophage-like Cells Identifies ARTD8/PARP14 and ARTD9/PARP9 ADP-Ribosylation

doi: 10.1021/acs.jproteome.8b00895

Figure Lengend Snippet: GPS leads to the identification of ADP-ribosylated ARTDs/PARPs other than ARTD1/PARP1. (A) A comparison using Venn diagrams for ADPr peptides found in two replicates for full scan (400–1500 m / z ) and combined 4× GPS scans (GPS-1, 400–605; GPS-2, 595–805; GPS-3, 795–1005; GPS-4, 995–1200 m / z ). (B) A comparison of ADPr peptides found in control and IFN-γ-treated THP-1 cells for the full scan and combined 4× GPS scans. (C) Sequence motif analysis for ADPr acceptor amino acids (N, number of ADPr peptides used for the analysis). (D) A plot of the number of ADP-ribosylation sites per protein. (E) Comparison of ADPr peptide abundances between control and IFN-γ in each replicate; regression lines, 95% confidence interval, and standard error of estimate (SEE) are provided (red dots are outliers). (F) MS/MS spectra of an ARTD8/PARP14 ADPr peptide using PRM acquisitions. Black peaks were manually annotated. *, ADPr site. (G) A comparison of the number of proteins identified in the Af1521 elution (ADPr proteins) and input samples (backbone proteins) per replicate. (H) A comparison of the relative changes to ADPr peptides versus their backbone proteins in response to IFN-γ (IFN-γ/control).

Article Snippet: Cell lysate from three 10 cm-dishes (1.0 mg) was incubated with 150 μL of Af1521 macrodomain conjugated to Glutathione sepharose 4B (GE Healthcare, Chicago, IL, Cat# 17075601) for 90 min at 4 °C using a rotator.

Techniques: Sequencing, Tandem Mass Spectroscopy

Journal: Journal of Proteome Research

Article Title: A Study into the ADP-Ribosylome of IFN-γ-Stimulated THP-1 Human Macrophage-like Cells Identifies ARTD8/PARP14 and ARTD9/PARP9 ADP-Ribosylation

doi: 10.1021/acs.jproteome.8b00895

Figure Lengend Snippet: IFN-γ increased ARTD8/PARP14 and ARTD9/PARP9 ADP-ribosylation. (A) A pan ADP-ribose Western blot analysis of control and IFN-γ-treated THP-1 cells after 10H IP or incubation with IgG. (B) A comparison of enriched proteins using a Venn diagram between 10H and IgG. The plot showing the log 2 (abundance ratio (10H/IgG)) and −log( p -value) of 551 shared proteins (Venn diagram). 114 proteins passed the threshold of abundance ratio (10H/IgG) > 10-fold and p -value < 0.05. (C) Abundance ratio (IFN-γ/control) of 10H-enriched proteins, ARTD9/PARP9, DNAJC13, and ITGB5, were unique to IFN-γ and filled with maximum. (D) Western blot analysis of Af1521- and 10H antibody-enriched ARTD/PARP enzymes from control, IFN-γ-treated, and PJ34 plus IFN-γ-treated THP-1 cells. (E) Comparing ADP-ribosylome from Af1521 (ADPr proteins) and 10H (backbone proteins) data set and showing the list of 39 common proteins between the two data sets.

Article Snippet: Cell lysate from three 10 cm-dishes (1.0 mg) was incubated with 150 μL of Af1521 macrodomain conjugated to Glutathione sepharose 4B (GE Healthcare, Chicago, IL, Cat# 17075601) for 90 min at 4 °C using a rotator.

Techniques: Western Blot, Incubation

Journal: Journal of Proteome Research

Article Title: A Study into the ADP-Ribosylome of IFN-γ-Stimulated THP-1 Human Macrophage-like Cells Identifies ARTD8/PARP14 and ARTD9/PARP9 ADP-Ribosylation

doi: 10.1021/acs.jproteome.8b00895

Figure Lengend Snippet: Protein–protein interactions (PPI) in IFN-γ-induced ADP-ribosylome on THP-1 cells. (A) A PPI network mapping all 145 ADPr proteins from Af1521 data and visualizing their interactions (confidence interaction scores ≥700). ADPr proteins with at least one interaction with another ADPr proteins are shown. Selected GO biological processes (GO-1 to GO-7 below) from the extended list in Table S4 . (B) PPI networks of 2-fold increased or decreased ADPr proteins in the IFN-γ compared to control. (C) PPI networks from a subset of 2-fold increasing or decreasing enriched proteins using the 10H workflow in response to IFN-γ stimulation.

Article Snippet: Cell lysate from three 10 cm-dishes (1.0 mg) was incubated with 150 μL of Af1521 macrodomain conjugated to Glutathione sepharose 4B (GE Healthcare, Chicago, IL, Cat# 17075601) for 90 min at 4 °C using a rotator.

Techniques: