n boc 1 4 butanediamine  (Chem Impex International)

Journal: Nucleic Acids Research

Article Title: Mono-ubiquitination of TopBP1 by PHRF1 enhances ATR activation and genomic stability

doi: 10.1093/nar/gkaf073

Figure Lengend Snippet: PHRF1 is recruited to replication forks. ( A ) Flag-PHRF1 overexpressed U2OS cells were stained with anti-Flag (PHRF1) and γH2AX antibodies 10 min post micro-irradiation. Scale bars, 10 μm. ( B , C ) PHRF1 and γH2AX colocalization were evaluated by immunofluorescence assay. The graphs in panels (B, C) represent mean ± standard error of mean, two-tailed, unpaired t -test; n = 4 independent experiments. Scale bars, 10 μm. ( D , E ) Cell cycle distribution was determined by flow cytometry in PHRF1 knockout or WT HCT116 cells. The proportions of cells in each cell cycle phase were determined using Modifit. The data in panel (D) are presented as mean ± standard error of mean, with statistical significance assessed using a two-tailed, unpaired t -test ( n = 3 independent experiments). PHRF1 knockout efficiency in panel (D) was evaluated using WB (panel E). ( F ) Cartoon scheme for iPOND assay. Image created in BioRender. Chenghui, C. (2025). https://BioRender.com/r26o549 . ( G ) HCT116 cells expressing Flag-PHRF1 were incubated with 10 μM EdU. Replication fork proteins were isolated by native-iPOND. ( H ) HCT116 cells were preincubated with 50 nM chaetocin, followed by incubated with 10 μM EdU and 10 mM HU for the indicated durations. Replication fork proteins were then isolated by native-iPOND. ( I ) U2OS cells were pre-treated with 50 nM chaetocin for 1 h, followed by micro-irradiation. The cells were then costained with anti-PHRF1 and γH2AX antibodies 10 min after micro-irradiation. Scale bars, 10 μm. ( J ) Biotin-labeled histone peptides were incubated with the indicated 293T cell lysates, captured with streptavidin beads, and analyzed by SDS–PAGE. ( K ) 293T cells were transfected with Flag-PHRF1 constructs for 48 h, then pretreated with or without 50 nM chaetocin for 1 h, followed by treatment with 10 mM HU for 2 h. The chromatin fraction was immunoprecipitated using Flag antibody-conjugated beads, and H3K9me3 and Flag-PHRF1 were detected by immunoblotting with the indicated antibodies. ( L ) 293T cells were transfected with the indicated Flag-PHRF1 constructs for 48 h. The chromatin fraction was immunoprecipitated with Flag antibody-conjugated beads. H3K9me3 and Flag were immunoblotted with the indicated antibodies. ( M ) 293T cells were treated with or without 10 mM HU for 2 h. Whole cell lysates were immunoprecipitated with PHRF1 antibody and rabbit IgG. TopBP1, p-S/TQ, and PHRF1 were blotted with the indicated antibodies. ( N ) 293T cells were transfected with Flag-PHRF1 for 48 h, followed by pretreatment with 1 μM VE-822 for 1 h. After VE-822 treatment, cells were incubated with 10 mM HU for 1 h. Whole cell lysates were immunoprecipitated using Flag antibody-conjugated beads. Flag and p-S/TQ were blotted with the indicated antibodies. ( O ) 293T cells were transfected with HA-PHRF1 WT, S925A, S1389A, or 2SA (S925A/S1389A) for 48 h, followed by treatment with 10 mM HU for 1 h. Whole cell lysates were immunoprecipitated using HA antibody-conjugated beads. HA and p-S/TQ were blotted with the indicated antibodies. ( P ) HCT116 cells were transfected with HA-PHRF1 or HA-PHRF1 S925A for 48 h and then incubated with 10 μM EdU and 10 mM HU for 2 h. Replication fork proteins were isolated by native-iPOND. HA and H3 were immunoblotted with the indicated antibodies.

Article Snippet: pcDNA3-LacR-TopBP1 (#31317) vector was purchased from Addgene.

Techniques: Staining, Irradiation, Immunofluorescence, Two Tailed Test, Flow Cytometry, Knock-Out, Expressing, Incubation, Isolation, Labeling, SDS Page, Transfection, Construct, Immunoprecipitation, Western Blot

Journal: Nucleic Acids Research

Article Title: Mono-ubiquitination of TopBP1 by PHRF1 enhances ATR activation and genomic stability

doi: 10.1093/nar/gkaf073

Figure Lengend Snippet: PHRF1 is important for ATR-Chk1 activation. ( A ) 293T cells were transfected with Flag-PHRF1 for 48 h. Whole cell lysate was immunoprecipitated with Flag antibody-conjugated beads and blotted with the indicated antibodies. ( B ) 293T cells were transfected with HA-PHRF1 WT or S925A mutant for 48 h, followed by treatment with or without 10 mM HU for 2 h. Chromatin fraction was immunoprecipitated with HA antibody-conjugated beads and blotted with the indicated antibodies. ( C ) 293T cells were transfected with Flag-PHRF1 WT, ΔRING, or ΔPHD for 48 h. Whole cell lysate was immunoprecipitated with Flag antibody-conjugated beads and blotted with the indicated antibodies. ( D ) Schematic representation of SFP-TopBP1 WT and truncation mutants. 293T cells were transfected with SFP-TopBP1 WT or SFP-TopBP1 mutants for 48 h. Whole cell lysate was immunoprecipitated with Flag antibody-conjugated beads and blotted with the indicated antibodies. ( E ) U2OS cells were infected with lentivirus expressing control shRNA or PHRF1 shRNA. The cells were treated with or without 10 mM HU for 2 h. Phospho-RPA32 (S33) foci formation was assessed by immunofluorescence, and the percentage of phospho-RPA32 (S33) foci > 5 cells [phospho-RPA32 (S33) positive cells] was quantified and plotted. The graph represents mean ± standard error of mean, two-tailed, unpaired t -test; n = 5 independent experiments. Scale bars, 10 μm. ( F ) PHRF1 knockout and WT HCT116 cells were treated with or without 10 mM HU for 2 h, and DNA damage-related proteins were assessed by immunoblotting. ( G ) PHRF1 knockout HCT116 cells were transfected with Flag-PHRF1 WT, ΔRING, or ΔPHD for 48 h, followed by treatment with or without 10 mM HU for 2 h. The indicated DNA damage-related proteins were then assessed by immunoblotting. t-Chk1: total Chk1, t-RPA32: total RPA32. p-chk1: phosphorylated chk1 (S345), and p-RPA32: phosphorylated RPA32 (S33). *indicates nonspecific bands.

Article Snippet: pcDNA3-LacR-TopBP1 (#31317) vector was purchased from Addgene.

Techniques: Activation Assay, Transfection, Immunoprecipitation, Mutagenesis, Infection, Expressing, Control, shRNA, Immunofluorescence, Two Tailed Test, Knock-Out, Western Blot

Journal: Nucleic Acids Research

Article Title: Mono-ubiquitination of TopBP1 by PHRF1 enhances ATR activation and genomic stability

doi: 10.1093/nar/gkaf073

Figure Lengend Snippet: PHRF1 mono-ubiquitinates TopBP1 at K73. ( A ) WT or PHRF1 knockout U2OS cells were treated with or without 10 mM HU for 2 h. TopBP1 foci formation was assessed by immunofluorescence and the percentage of cells with > 5 TopBP1 foci (TopBP1 positive cells) was quantified and plotted. The graphs represent mean ± standard error of mean, two-tailed, unpaired t -test; n = 3 independent experiments. Scale bars, 10 μm. ( B ) PHRF1 knockout and WT HCT116 cells were treated with 10 mM HU for 2 h. Chromatin fraction was isolated and immunoblotted using the indicated antibodies. ( C , D ) HCT116 cells were transfected with His-Ub and Flag-PHRF1 vector for 48 h. The cells were then treated either with vehicle (C) or 10 mM HU for 2 h (D), followed by lysis in urea denaturation buffer. The ubiquitination of TopBP1 was assessed by His pull-down, and the samples were immunoblotted with the indicated antibodies. ( E ) PHRF1 knockout and WT HCT116 cells were transfected with His-Ub vector for 48 h, followed by a His pull-down assay. ( F ) PHRF1 knockout HCT116 cells were transfected with Flag-PHRF1 WT, CA, or S925A mutant and GFP-TopBP1 vectors for 48 h, followed by a His pull-down assay. ( G ) HCT116 cells were transfected with His-Ub, HA-PHRF1, GFP-TopBP1 WT, and different KR mutant vectors for 48 h, followed by a His pull-down assay. (E – G) The cells were lysed using a urea denaturation buffer. The ubiquitination of TopBP1 was assessed by His pull-down, and the samples were immunoblotted with the indicated antibodies.

Article Snippet: pcDNA3-LacR-TopBP1 (#31317) vector was purchased from Addgene.

Techniques: Knock-Out, Immunofluorescence, Two Tailed Test, Isolation, Transfection, Plasmid Preparation, Lysis, Pull Down Assay, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Mono-ubiquitination of TopBP1 by PHRF1 enhances ATR activation and genomic stability

doi: 10.1093/nar/gkaf073

Figure Lengend Snippet: PHRF1 promotes the interaction between TopBP1 and ATR. ( A – C ) The indicated vectors were transfected into PHRF1 knockout or WT HCT116 cells. 48 h later, the cells were then treated either with vehicle or 10 mM HU for 2 h. The interaction between ATR and TopBP1 was determined by immunoprecipitation using the indicated antibodies. ( D ) HCT116 cells were transfected with S-TopBP1 C-terminus and Flag-TopBP1 N-terminus vectors for 48 h. TopBP1 N-terminus and C-terminus interaction was determined by immunoprecipitation assay using S beads. ( E ) HCT116 cells were transfected with Flag-TopBP1 N-terminus and S-TopBP1 C-terminus vectors for 48 h. The dose of N-terminus of TopBP1 vector for transfection ranges from 0.5 to 4 μg in the indicated groups, while the dose of C-terminus is 1 μg in each group. Immunoprecipitation and immunoblotting were conducted using the indicated antibodies. ( F , G ) PHRF1 knockout or WT U2OS cells were transfected with the indicated vectors. 48 h later, the cells were treated either with vehicle or 10 mM HU for 2 h as indicated in each group. The interaction between TopBP1 N-term and C-term was assessed by PLA. PLA foci are shown in panel (F), and the percentage PLA signal positive cells (≥ 1 PLA foci) were quantified in the graph in panel (G). Eight hundred cells were quantified for each independent experiment, n = 3 independent experiments. The graph represents mean ± standard error of mean, two-tailed, unpaired t -test. Scale bars, 10 μm. ( H ) The graphic summary of TopBP1 regulation by PHRF1.

Article Snippet: pcDNA3-LacR-TopBP1 (#31317) vector was purchased from Addgene.

Techniques: Transfection, Knock-Out, Immunoprecipitation, Plasmid Preparation, Western Blot, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Mono-ubiquitination of TopBP1 by PHRF1 enhances ATR activation and genomic stability

doi: 10.1093/nar/gkaf073

Figure Lengend Snippet: PHRF1 promotes stalled replication fork restart by ubiquitinating TopBP1. ( A – E ) WT or PHRF1 knockout HCT116 cells were transfected with the indicated vectors or siRNAs. 48 h later, cells were labeled with 25 μM IdU, followed by 200 μM CldU in each experiment. Cells were incubated with IdU for 20 min and then treated either with vehicle in panels (A,D) or 4 mM HU in panels (B, C, and E) for 2 h. After incubation, cells were labeled with CldU for 20 min. DNA fibers were stretched on a microscope slide, stained with IdU and CldU antibodies, and imaged. Fork speed was determined by measuring the length of the CldU track in panels (A, D) ( n = 100). Stalled replication fork restart was assessed by measuring the length of the CldU track in panels (B, C, and E) ( n = 100). ( F , G ) PHRF1 knockout or WT HCT116 cells were treated with 2 mM HU for 24 h, followed by release at the indicated times. Cells were collected, and γH2AX-positive in panel (F) and subG1 population (an indicator of cell death) in panel (G) were determined by flow cytometry. ( H – K ) PHRF1 knockout or WT HCT116 cells were transfected with the indicated vectors and then seeded into 6-well plates. Cells were treated with the indicated doses of HU (H), CPT (I), 5-FU (J), or UV (K). After 14 days, colony formation was assessed and the number of colonies was counted. The graphs represent mean ± standard error of mean, two-tailed, unpaired t -test; n = 3 independent experiments.

Article Snippet: pcDNA3-LacR-TopBP1 (#31317) vector was purchased from Addgene.

Techniques: Knock-Out, Transfection, Labeling, Incubation, Microscopy, Staining, Flow Cytometry, Two Tailed Test

Journal: World Journal of Microbiology & Biotechnology

Article Title: Fungicide sensitivity of grapevine bacteria with plant growth-promoting traits and antagonistic activity as non-target microorganisms

doi: 10.1007/s11274-023-03569-5

Figure Lengend Snippet: Identification of 58 isolates isolated from grapevine by alignment of 16 S rRNA gene sequence against sequence provided by the EzTaxon-E database

Article Snippet: , Curtobacterium oceanosedimentum ATCC 31,317(T) [EF592577] , , 100.

Techniques: Isolation, Sequencing

Journal: World Journal of Microbiology & Biotechnology

Article Title: Fungicide sensitivity of grapevine bacteria with plant growth-promoting traits and antagonistic activity as non-target microorganisms

doi: 10.1007/s11274-023-03569-5

Figure Lengend Snippet: Plant growth promoting (phosphate solubilization using NBRIP and PVK media supplemented with Ca 3 (PO 4 ) 2 or CaHPO 4 , and siderophores production) traits and antagonistic activity of 58 bacteria isolated from grapevines against Aspergillus niger An3 (An) and Botrytis cinerea ITEM 1719 (Bc). Activities were estimated measuring the radius of clear (phosphate solubilization and antagonism) or colored (siderophores) zone around colony as (−) negative, (+/−) weakly positive, (+) positive and (++) strongly positive

Article Snippet: , Curtobacterium oceanosedimentum ATCC 31,317(T) [EF592577] , , 100.

Techniques: Activity Assay, Bacteria, Isolation

Journal: World Journal of Microbiology & Biotechnology

Article Title: Fungicide sensitivity of grapevine bacteria with plant growth-promoting traits and antagonistic activity as non-target microorganisms

doi: 10.1007/s11274-023-03569-5

Figure Lengend Snippet: Results of plate assay to evaluate the sensitivity of 100 isolates (+ sensitive, – resistant) to seven commercial fungicides, Dedalus® (Ded.), Lidal® (Lid.), Topas® (Top.), Switch® (Swi.), Tucana® (Tuc.), Carson® (Car.) and Folpan® (Fol.)

Article Snippet: , Curtobacterium oceanosedimentum ATCC 31,317(T) [EF592577] , , 100.

Techniques: