rabbit anti polλ  (Bethyl)

Journal: Mutagenesis

Article Title: Mutations induced by 8-oxo-7,8-dihydroguanine in WRN- and DNA polymerase λ-double knockdown cells.

doi: 10.1093/mutage/gey024

Figure Lengend Snippet: Fig. 2. Effects of the WRN and DNA pol λ double knockdown in U2OS cells on the mutant frequency induced by GO. Open columns, control plasmid containing G at position 122; closed columns, plasmid containing GO at position 122. Transfection experiments were performed six times. Data are expressed as the means + standard errors. *P < 0.05 vs. control RNA (Student’s t-test).

Article Snippet: To detect DNA pol λ, the membranes were blocked in Blocking One (Nacalai Tesque, Kyoto, Japan) for 1 h at room temperature, and then incubated with a rabbit anti-DNA pol λ antibody (Bethyl Laboratories, Montgomery, TX, USA, catalogue no. A301-640A) in PBS-T containing 5% Blocking One overnight at 4°C.

Techniques: Knockdown, Mutagenesis, Control, Plasmid Preparation, Transfection

Journal: Mutagenesis

Article Title: Mutations induced by 8-oxo-7,8-dihydroguanine in WRN- and DNA polymerase λ-double knockdown cells.

doi: 10.1093/mutage/gey024

Figure Lengend Snippet: Fig. 1. Knockdowns of WRN and DNA pol λ by siRNAs. U2OS cells were treated with the siRNAs, and total protein was extracted at 24, 48 and 72 h after siRNA introduction. WRN and DNA pol λ expressions were analysed by western blotting.

Article Snippet: To detect DNA pol λ, the membranes were blocked in Blocking One (Nacalai Tesque, Kyoto, Japan) for 1 h at room temperature, and then incubated with a rabbit anti-DNA pol λ antibody (Bethyl Laboratories, Montgomery, TX, USA, catalogue no. A301-640A) in PBS-T containing 5% Blocking One overnight at 4°C.

Techniques: Western Blot

Journal: Nature communications

Article Title: PAXX and its paralogs synergistically direct DNA polymerase λ activity in DNA repair.

doi: 10.1038/s41467-018-06127-y

Figure Lengend Snippet: Fig. 3 Pol λ interacts with XRCC4 family proteins via its BRCT domain in cells. a HEK293F cells were irradiated with 10 Gy X-ray or left untreated. Soluble nuclear extracts were isolated following 0–60 min post-irradiation recovery time at 37 °C. Following IP with anti-Pol λ or rabbit IgG (rIgG), Pol λ and associated proteins were resolved by SDS-PAGE and immunoblotted for the indicated NHEJ factors. b HEK293F cell nucleoplasmic (NP) or soluble chromatin (sol. Chr) extracts were immunoprecipitated with rIgG, anti-PAXX or -XLF or mouse IgG (mIgG) or anti-XRCC4. Immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotted for the indicated NHEJ factors. c As described in Panel A, except that soluble nuclear extracts were incubated with 0-200 μg/ml EtBr for 1 h prior to IP with anti-Pol λ or rIgG. d EMSA showing that interaction of Pol λ with DNA-bound Ku requires R57 and L60 in the BRCT domain of Pol λ. Reactions were performed with IRDye® 700-labelled 5nt-gapped dsDNA (33-mer) in the presence or absence of FLAG- Ku70/80 (20 nM) and either FLAG-Pol λ-WT or a R57A/L60A mutant (50 nM). e HEK293F cells were transiently transfected with either pCMX-LacZ (control) or pCMX-FLAG-Pol λ-WT, -ΔBRCT or a R57A/L60A mutant and anti-FLAG IPs performed

Article Snippet: Assays were also performed with endogenous Pol λ immunoprecipitated from RPE-1 cells using anti-rabbit Pol λ (Bethyl A301-640A).

Techniques: Irradiation, Isolation, SDS Page, Immunoprecipitation, Incubation, Mutagenesis, Transfection, Control

Journal: Nature communications

Article Title: PAXX and its paralogs synergistically direct DNA polymerase λ activity in DNA repair.

doi: 10.1038/s41467-018-06127-y

Figure Lengend Snippet: Fig. 4 Interaction of PAXX and XLF with Pol λ requires C-terminal Ku-binding regions. a EMSA showing that interaction of PAXX with Pol λ requires DNA- bound Ku. Reactions were performed with 10 or 20 nM IRDye® 700-labelled 5nt-gapped dsDNA (90-mer) and the following concentrations of FLAG- Ku70/80 (20 nM), FLAG-Pol λ (40 nM) or cleaved PAXX (100 nM). b Binding of PAXX to DNA-bound Ku requires C-terminal residues V199 and F201 of PAXX. Reactions were performed with 20 nM IRDye® 700-labelled 5nt-gapped dsDNA (90-mer) and the indicated concentrations of FLAG-PAXX-WT or a FLAG-PAXX-V199A/F201A mutant and FLAG-Ku70/80 (20 nM). c As described in Panel A, except that reactions contained FLAG-Ku (20 nM), FLAG-Pol λ (100 nM), FLAG-PAXX-WT (2.5 μM, left panel) or a V199A/F201A mutant (2.5 μM, right panel). d As described in Panel A, except that reactions contained FLAG-Ku (20 nM), FLAG-Pol λ (200 nM), FLAG-XLF-WT (2.5 μM, left panel) or a C-terminal FLAG-XLF (aa1-233) deletion mutant (2.5 μM, right panel)

Article Snippet: Assays were also performed with endogenous Pol λ immunoprecipitated from RPE-1 cells using anti-rabbit Pol λ (Bethyl A301-640A).

Techniques: Binding Assay, Mutagenesis

Journal: Nature communications

Article Title: PAXX and its paralogs synergistically direct DNA polymerase λ activity in DNA repair.

doi: 10.1038/s41467-018-06127-y

Figure Lengend Snippet: Fig. 5 Role of XRCC4 family members in the recruitment of Pol λ to laser microirradiation-induced DNA damage sites. a Upper, Schematic figure showing N-terminal EGFP- and mCherry-Pol λ fusion proteins; Lower, Representative immunofluorescence images showing that N-terminal EGFP- and mCherry-Pol λ fusion proteins are localised to nuclei in U2OS cells. b Recruitment of N-terminal EGFP-Pol λ to laser-induced DNA damage sites in U2OS cells. c Time course of N-EGFP-Pol λ recruitment to laser-induced DNA damage sites in U2OS cells. Data shown are the mean and SEM from 16 individual cells. d Immunoblot analysis of N-EGFP-Pol λ expressing U2OS cells deficient in PAXX, XLF or XRCC4 generated by CRISPR-Cas9. WCL were resolved by SDS- PAGE and the indicated proteins detected by immunoblotting. e Localisation of N-mCherry-Pol λ in U2OS WT, PAXX-, XLF- and XRCC4-deficient cells. Representative immunofluorescence images showing that N-terminal mCherry-Pol λ fusion protein localises to nuclei in PAXX-, XLF- or XRCC4-deficient U2OS cells. Cells were co-stained with DAPI or dynamin-2, a perinuclear-enriched protein. f Time course of N-EGFP-Pol λ recruitment to laser-induced DNA damage sites in U2OS-WT cells and cells deficient in PAXX, XLF or XRCC4. Data shown are the mean and SEM from WT, PAXX, XLF and XRCC4 knockout cells. Graphs shown are for the following cell numbers: WT: 8 cells; PAXX KO, 10 cells; XLF KO 17 cells; XRCC4 KO 18 cells. g Time course of N-EGFP-FLAG-Ku70 recruitment to laser-induced DNA damage sites in U2OS-WT cells and PAXX KO cells. Data shown are the mean and SEM from WT and PAXX knockout cells. Graphs shown are for the following cell numbers: WT: 17 cells; PAXX KO, 19 cells

Article Snippet: Assays were also performed with endogenous Pol λ immunoprecipitated from RPE-1 cells using anti-rabbit Pol λ (Bethyl A301-640A).

Techniques: Western Blot, Expressing, Generated, CRISPR, SDS Page, Staining, Knock-Out

Journal: Nature communications

Article Title: PAXX and its paralogs synergistically direct DNA polymerase λ activity in DNA repair.

doi: 10.1038/s41467-018-06127-y

Figure Lengend Snippet: Fig. 6 XRCC4 family proteins stimulate gap filling synthesis activity of Pol λ. a Gap filling activity of Pol λ-WT and a catalytically inactive Pol λ-D427A/ D429A/D490A mutant. b PAXX, XLF and XRCC4 stimulate gap-filling synthesis activity of Pol λ with an IRDye® 700-labelled 5nt-gapped dsDNA (33- mer) substrate. c As described in Panel B, except that some reactions also contained either FLAG-PAXX or –XLF alone d Gap filling synthesis assays were performed as described in Panel B with Pol λ immunoprecipitated from RPE-1 PAXX+/+ or PAXX KO cells incubated with or without XLF or XRCC4 siRNA

Article Snippet: Assays were also performed with endogenous Pol λ immunoprecipitated from RPE-1 cells using anti-rabbit Pol λ (Bethyl A301-640A).

Techniques: Activity Assay, Mutagenesis, Immunoprecipitation, Incubation

Journal: Nature communications

Article Title: PAXX and its paralogs synergistically direct DNA polymerase λ activity in DNA repair.

doi: 10.1038/s41467-018-06127-y

Figure Lengend Snippet: Fig. 7 Head domains of XRCC4 Family Proteins interact with and stimulate Pol λ-dependent gap filling synthesis. a Schematic representation of XRCC4 family proteins. b PAXX head domain, but not the CC-CTR region, stimulates gap filling synthesis activity of Pol λ. c Silver stain and immunoblot analysis of purified XRCC4 family protein head domains. d Head domain of XRCC4 family proteins stimulate Pol λ-dependent gap filling synthesis activity. e Far- Western blot analysis showing that Pol λ-WT interacts with the head domain of XRCC4 family proteins. f Stimulation of Pol λ-dependent gap filling synthesis activity by PAXX-WT but not PAXX-VF, a non-Ku binding C-terminal mutant. g PAXX head domain stimulates comparable gap filling synthesis activity of Pol λ-WT and –R57A/L60A

Article Snippet: Assays were also performed with endogenous Pol λ immunoprecipitated from RPE-1 cells using anti-rabbit Pol λ (Bethyl A301-640A).

Techniques: Activity Assay, Silver Staining, Western Blot, Far Western Blot, Binding Assay, Mutagenesis

Journal: Nature communications

Article Title: PAXX and its paralogs synergistically direct DNA polymerase λ activity in DNA repair.

doi: 10.1038/s41467-018-06127-y

Figure Lengend Snippet: Fig. 8 The Pol λ 8 kDa domain is required for stimulation of Pol λ-dependent gap filling activity via interaction with the head domain of XRCC4 family proteins. a Schematic representation of N-terminal Pol λ deletion mutants. b PAXX, XLF and XRCC4 stimulate gap filling synthesis activity of ΔBRCT- and ΔBRCT-Ser-Pro-Pol λ but not ΔBRCT-Ser-Pro-8kDa-Pol λ with an IRDye® 700-labelled 5nt-gapped dsDNA (33-mer) substrate. c PAXX head domain promotes gap filling synthesis activity of ΔBRCT- and ΔBRCT-Ser-Pro-Pol λ but not ΔBRCT-Ser-Pro-8kDa-Pol λ. d Far-Western blot analysis showing that the head domain of XRCC4 family proteins interacts with Pol λ-WT and -ΔBRCT but not -ΔBRCT-Ser-Pro-8kDa

Article Snippet: Assays were also performed with endogenous Pol λ immunoprecipitated from RPE-1 cells using anti-rabbit Pol λ (Bethyl A301-640A).

Techniques: Activity Assay, Far Western Blot

Journal: Nature communications

Article Title: PAXX and its paralogs synergistically direct DNA polymerase λ activity in DNA repair.

doi: 10.1038/s41467-018-06127-y

Figure Lengend Snippet: Fig. 9 PAXX and XLF together with Pol λ to promote ligation of noncohesive DNA ends which requires gap filling activity of Pol λ. a–e Linear DNA substrates as shown were incubated with the indicated combinations of XRCC4/Lig IV, Ku70/80, PAXX, XLF and Pol λ and the joining efficiency quantified by qPCR with a TaqMan probe using a standard curve of log10 % joining efficiency versus Ct value generated using prejoined DNA fragments. DNA substrates were as follows: (a) EcoRV-PvuI blunt-2nt 3’ overhang; (b) EcoRV-KpnI blunt-4nt 3′ overhang; (c) EcoRV-EcoRV blunt-blunt ends; (d) EcoRI- KpnI 4nt 5’ overhang-4nt 3′ overhang, (e) EcoRV-BstEII blunt-5nt 5′ overhang; (f) As described in Panel (e), except that ligation assays contained either Pol λ-WT or a catalytically inactive Pol λ-3D mutant. Results shown are the mean ± SEM from 2–3 experiments performed in triplicate

Article Snippet: Assays were also performed with endogenous Pol λ immunoprecipitated from RPE-1 cells using anti-rabbit Pol λ (Bethyl A301-640A).

Techniques: Ligation, Activity Assay, Incubation, Generated, Mutagenesis

Journal: Nature communications

Article Title: PAXX and its paralogs synergistically direct DNA polymerase λ activity in DNA repair.

doi: 10.1038/s41467-018-06127-y

Figure Lengend Snippet: Fig. 10 Pol λ, PAXX and XLF function in common and parallel pathways. a Immunoblot analysis of control- or Pol λ siRNA-depleted U2OS PAXX KO, XLF KO and PAXX/XLF DKO cells. WCL were resolved by SDS-PAGE and indicated proteins detected by immunoblotting. b Clonogenic survival assays following IR (0-4 Gy) for U2OS WT, -PAXX KO, -XLF KO and -PAXX/XLF DKO cells with or without depletion of Pol λ Mean and SD from three independent experiments are shown. Statistical analysis was performed using a two-tailed paired t-test to compare cells incubated with Pol λ siRNA with control siRNA: 1 Gy - WT p = 0.91, PAXX KO p = 0.38, XLF KO p = 0.10, PAXX/XLF DKO p = 0.10; 2 Gy - WT p = 0.0003, PAXX KO p = 0.0007, XLF KO p = 0.36, PAXX/XLF DKO p = 0.82; 4 Gy - WT = 0.02, PAXX KO = 0.002, XLF KO p = not determined, PAXX/XLF DKO p = not determined. c Cartoon showing a model for regulation of Pol λ by XRCC4 family proteins. At DSBs that are positioned proximal to a Pol λ substrate gap XRCC4 family proteins strongly interact with Ku heterodimers via their C-terminal regions; their head domains promote gap filling synthesis activity via comparatively weakly binding to the 8 kDa domain of Pol λ, which interacts with the 5′ end of the gap. Pol λ strongly interacts with Ku heterodimers via its N-terminal BRCT domain

Article Snippet: Assays were also performed with endogenous Pol λ immunoprecipitated from RPE-1 cells using anti-rabbit Pol λ (Bethyl A301-640A).

Techniques: Western Blot, Control, SDS Page, Two Tailed Test, Incubation, Activity Assay, Binding Assay