Anti-Ku80 抗体 [EPR3468]

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Ku80 antibody [EPR3468] (AB80592)

Immunofluorescence analysis of HeLa cells with 1/500 ab80592.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Ku80 antibody [EPR3468] (AB80592)

Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling Ku80 with unpurified ab80592 at 1/20 dilution (10ug/mL) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluorr^® 488) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

• IP

Unknown

Immunoprecipitation - Anti-Ku80 antibody [EPR3468] (AB80592)

ab80592 (purified) at 1/500 dilution (1.86 © : g/ml) immunoprecipitating Ku80 in HeLa whole cell lysate.
Lane 1 (input) : HeLa(Human cervix adenocarcinoma epithelial cell) whole cell lysate 10© : g
Lane 2 (+) : ab80592 & HeLa whole cell lysate
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab80592 in HeLa whole cell lysate
For western blotting, VeriBlot for IP secondary antibody (HRP) (ab131366) was used as the secondary antibody at 1/1000 dilution.
Blocking and diluting buffer : 5% NFDM /TBST .

All lanes:

Immunoprecipitation - Anti-Ku80 antibody [EPR3468] (ab80592)

Predicted band size: 83 kDa

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• WB

Lab

Western blot - Anti-Ku80 antibody [EPR3468] (AB80592)

All lanes:

Western blot - Anti-Ku80 antibody [EPR3468] (ab80592) at 1/3000 dilution

Lane 1:

PC-12(Rat adrenal gland pheochromocytoma) whole cell lysate at 20 µg

Lane 2:

NIH/3T3(Mouse embryonic fibroblast) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 83 kDa

Observed band size: 83 kDa

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• WB

Unknown

Western blot - Anti-Ku80 antibody [EPR3468] (AB80592)

All lanes:

Western blot - Anti-Ku80 antibody [EPR3468] (ab80592) at 1/50000 dilution

Lane 1:

A549 cell lysate at 10 µg

Lane 2:

HeLa cell lysate at 10 µg

Lane 3:

HepG2 cell lysate at 10 µg

Lane 4:

MCF7 cell lysate at 10 µg

Secondary

All lanes:

goat anti-rabbit HRP at 1/2000 dilution

Predicted band size: 83 kDa

Observed band size: 83 kDa

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• WB

CiteAb

Western blot - Anti-Ku80 antibody [EPR3468] (AB80592)

Western Blotting using Anti-Ku80 antibody [EPR3468], ab80592. Publication image from Rulten, S. L. et al., 2016, Nat Commun, 27063109. Legend direct from paper.

The WRN C-terminal KBM and XLF-like motif bind Ku protein complexes cooperatively.(a) HEK293T cells were co-transfected with expression constructs encoding GFP or the indicated GFP-tagged KBMs and GFP-tagged proteins recovered using GFP-TRAP beads. Aliquots of the input and eluate samples were fractionated by SDS–PAGE and immunoblotted for GFP, Ku80 and DNA–PKcs (CS). Right, cartoon depicting WRN and the position of the KBMs and XLF-like motif and the mutations employed in these experiments. (b) HEK293T cells were transfected with expression constructs encoding the indicated wild-type or mutated GFP-tagged WRN C-terminal KBM, XLF-like motif (‘X'), or KBM plus XLF-like motif in tandem. Cells were micro-irradiated with UVA as in Fig. 2. Representative images (left) and quantification (right) are shown. All quantified data are the mean GFP fluorescence (±s.e.m.) in the laser track relative to the mean GFP fluorescence before irradiation (set at 100%) from >20 cells per experiment. (c,d) Expression constructs encoding full-length wild-type (‘WT') GFP–WRN or derivatives harbouring the indicated point mutations in the N-terminal KBM (W18G), C-terminal KBM (W1410G) or deleted C-terminal tandem domain (δcAX) or XLF-like motif (δX ) were transfected into HEK293T cells and recovered using GFP-TRAP beads. Input and eluates were immunoblotted for GFP and Ku80. Numbers in parentheses are the fraction of Ku co-precipitated by the indicated GFP-tagged WRN protein, relative to wild-type WRN, quantified by ImageJ. Data are from two to six independent experiments, except for W18G/δcAX in which Ku recovery was too low to be determined (‘nd'). (e) Direct interaction of purified full-length Strep-tagged WRN with recombinant human Ku. Recombinant Strep-tagged WRN, WRNδcAX or WRNW18G was immobilized on Streptavidin Mag sepharose beads and incubated with recombinant Ku heterodimer. Aliquots of the recombinant proteins employed in the experiment are shown on the left (lanes 1–3) and proteins pulled down by the indicated Strep-tagged WRN protein are shown on the right (lanes 4–7). Lane 6 contains the proteins recovered in a control pull-down that lacked Strep-tagged WRN. Proteins were fractioned by SDS–PAGE and stained with Coomassie Blue.

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• WB

CiteAb

Western blot - Anti-Ku80 antibody [EPR3468] (AB80592)

Western Blotting using Anti-Ku80 antibody [EPR3468], ab80592. Publication image from Rulten, S. L. et al., 2016, Nat Commun, 27063109. Legend direct from paper.

The WRN C-terminal KBM and XLF-like motif bind Ku protein complexes cooperatively.(a) HEK293T cells were co-transfected with expression constructs encoding GFP or the indicated GFP-tagged KBMs and GFP-tagged proteins recovered using GFP-TRAP beads. Aliquots of the input and eluate samples were fractionated by SDS–PAGE and immunoblotted for GFP, Ku80 and DNA–PKcs (CS). Right, cartoon depicting WRN and the position of the KBMs and XLF-like motif and the mutations employed in these experiments. (b) HEK293T cells were transfected with expression constructs encoding the indicated wild-type or mutated GFP-tagged WRN C-terminal KBM, XLF-like motif (‘X'), or KBM plus XLF-like motif in tandem. Cells were micro-irradiated with UVA as in Fig. 2. Representative images (left) and quantification (right) are shown. All quantified data are the mean GFP fluorescence (±s.e.m.) in the laser track relative to the mean GFP fluorescence before irradiation (set at 100%) from >20 cells per experiment. (c,d) Expression constructs encoding full-length wild-type (‘WT') GFP–WRN or derivatives harbouring the indicated point mutations in the N-terminal KBM (W18G), C-terminal KBM (W1410G) or deleted C-terminal tandem domain (δcAX) or XLF-like motif (δX ) were transfected into HEK293T cells and recovered using GFP-TRAP beads. Input and eluates were immunoblotted for GFP and Ku80. Numbers in parentheses are the fraction of Ku co-precipitated by the indicated GFP-tagged WRN protein, relative to wild-type WRN, quantified by ImageJ. Data are from two to six independent experiments, except for W18G/δcAX in which Ku recovery was too low to be determined (‘nd'). (e) Direct interaction of purified full-length Strep-tagged WRN with recombinant human Ku. Recombinant Strep-tagged WRN, WRNδcAX or WRNW18G was immobilized on Streptavidin Mag sepharose beads and incubated with recombinant Ku heterodimer. Aliquots of the recombinant proteins employed in the experiment are shown on the left (lanes 1–3) and proteins pulled down by the indicated Strep-tagged WRN protein are shown on the right (lanes 4–7). Lane 6 contains the proteins recovered in a control pull-down that lacked Strep-tagged WRN. Proteins were fractioned by SDS–PAGE and stained with Coomassie Blue.

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• WB

CiteAb

Western blot - Anti-Ku80 antibody [EPR3468] (AB80592)

Western Blotting using Anti-Ku80 antibody [EPR3468], ab80592. Publication image from Rulten, S. L. et al., 2016, Nat Commun, 27063109. Legend direct from paper.

The WRN N-terminal KBM promotes WRN exonuclease activity.(a) Left, cartoon illustrating the GFP-tagged truncated recombinant WRN proteins employed in these experiments. The WRN N-terminal (‘nA') and C-terminal (‘cA') KBMs are indicated by red boxes and XLF-like motif (‘X') by a blue box. The exonuclease domain is indicated by a black box, and the position of the KBM mutation (W18G) by an asterisk and dotted line. Middle, U2-OS cells transiently expressing the indicated recombinant GFP-tagged WRN protein were imaged for GFP before and after UVA microirradiation, as in Fig. 2. Right, Ku80−/− MEFs transiently co-expressing GFP-tagged WRN-Exo, RFP-Ku70, and either RFP (vector), RFP-Ku80 or RFP-Ku80L68R as indicated were micro-irradiated as in Fig. 1. Data are the mean GFP fluorescence (±s.e.m.) in the laser track relative to the mean GFP fluorescence before irradiation (set at 100%) from >20 cells per experiment. (b) The indicated GFP-tagged WRN proteins were recovered from transiently transfected HEK293T cell lysates pre-treated or not as indicated with Benzonase and RNAse in pull-down assays using GFP-TRAP beads. Aliquots of the bead eluate were fractionated by SDS-PAGE and silver stained to detect GFP-WRN, GFP-WRNW18G, Ku80, and DNA-PKcs (‘CS'). (c) Cy3-labeled 30 bp duplex oligonucleotide (20 nM) with a 5′ overhang was incubated with 500, 100, 20 or 5 nM HIs-tagged WRN-Exo or WRN-ExoW18G in the absence or presence of 100 nM Ku heterodimer (Ku70/Ku80, ‘Ku') and 5 mM MgCl2. Exonuclease products were resolved on a 16% TBE-Urea gel. (d) Exonuclease assays were conducted as above in the presence of 5 mM MgCl2 using 10 nM His-tagged WRN-Exo and 100, 20, 4 or 0.8 nM of either Ku heterodimer (Ku70/Ku80; ‘Ku'), KuδC heterodimer (Ku70/Ku80δC; ‘KuδC'), or mutant KuδC heterodimer harbouring the Ku80 mutation, L68R (KuδCL68R). (e) Exonuclease assays were conducted as above using 100, 20 and 4 nM of the indicated His-tagged WRN protein and 10 nM wild-type Ku heterodimer (Ku70/Ku80; ‘Ku') in 5 mM Mg2+.

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