Anti-DDB1 抗体 [EPR6089]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DDB1 antibody [EPR6089] (AB109027)

Immunohistochemical analysis of paraffin-embedded Human breast tissue using ab109027 at a dilution of 1/100. Antigen retrieval was heat mediated before commencing with IHC staining protocol.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-DDB1 antibody [EPR6089] (AB109027)

ab109027 stained UV-treated HeLa cells. The cells were 4% formaldehyde fixed for 10 minutes at room temperature and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1hour at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab109027 at 1/100 dilution) overnight at +4°C. The secondary antibody (pseudo-colored green) was ab150081 used at a 1/1000 dilution for 1hour at room temperature. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43μM for 1hour at room temperature.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-DDB1 antibody [EPR6089] (AB109027)

Immunocytochemistry analysis of NIH/3T3 (mouse embryonic fibroblast) cells labeling DDB1 with ab109027 at 1/250 (8.9 μg/mL). Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% tritonX-100. ab150077 AlexaFluor^®488 Goat anti-Rabbit at 1/1000 (2 μg/mL) was used as the secondary antibody. DAPI (blue) was used as nuclear counterstain.

Confocal image showing nuclear and cytoplasmic staining in NIH/3T3 cells.

• WB

Lab

Western blot - Anti-DDB1 antibody [EPR6089] (AB109027)

This blot was produced using 4-20% SDS-PAGE containing 15 μg of HeLa whole cell lysate per lane at 150V for 1hr before being transferred onto a 0.45 μm PVDF membrane at 75V for 1hr. The membrane was then blocked for 1hr using 5% NFDM/TBST, then incubated with ab109027 (1/50,000) at room temperature for 1hr. After being washed three times in TBST, the membrane was incubated with Peroxidase conjugated goat anti-rabbit IgG (H+L) (ab97051) at 1/20,000 dilution for 1hr at room temperature. The membrane was washed three times again. Then the signal was developed using the ECL technique ab109027 was stored at a range of temperatures (+4°C, +22°C, +37°C) for 1 week before being tested in WB. The image shows the band intensity remains relatively constant across all storage temperatures, demonstrating that antibody activity is not affected.

All lanes:

Western blot - Anti-DDB1 antibody [EPR6089] (ab109027) at 1/50000 dilution

All lanes:

HeLa whole cell lysate at 15 µg with NDFM/TBST

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

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Exposure time: 10s

• WB

Unknown

Western blot - Anti-DDB1 antibody [EPR6089] (AB109027)

All lanes:

Western blot - Anti-DDB1 antibody [EPR6089] (ab109027) at 1/50000 dilution

Lane 1:

HepG2 cell lysate at 10 µg

Lane 2:

HeLa cell lysate at 10 µg

Lane 3:

NIH3T3 cell lysate at 10 µg

Lane 4:

Human platelet lysate at 10 µg

Predicted band size: 127 kDa

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• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-DDB1 antibody [EPR6089] (AB109027)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.

• WB

CiteAb

Western blot - Anti-DDB1 antibody [EPR6089] (AB109027)

Western Blotting using Anti-DDB1 antibody [EPR6089], ab109027. Publication image from Lu, H. et al., 2017, Nat Commun, 29229926. Legend direct from paper.

DDB1-CUL4A E3 ubiquitin ligase stimulates RECQL4 recruitment to DSBs. a Co-IP analysis for the interaction of RECQL4 with DDB1 and CUL4A in HEK293T cells. DDB1 and CUL4A were pulled down with 3xFLAG-tagged RECQL4 from HEK293T cells. b N-terminal domain of RECQL4 interacts with DDB1 in HEK293T cells. Upper panel shows schematic diagram of 3xFLAG-tagged RECQL4 and the truncated fragments. N-terminal domain (RQ4-NT), helicase domain (RQ4-HE), and C-terminal domain (RQ4-CT) were tagged with both 3xFLAG and SV40 nuclear-location sequence (NLS). c Knockdown of either DDB1 or CUL4A inhibits ubiquitination of RECQL4 in U2OS cells. Endogenous RECQL4 was purified with anti-RECQL4 antibody from denatured U2OS cell lysates and the ubiquitination was then detected with anti-Ub antibody. d Inhibition of CDK1 and CDK2 reduced the ubiquitination of RECQL4 in U2OS cells. RECQL4 was immunoprecipitated with anti-RECQL4 antibody from U2OS cells, and its ubiquitination was examined with anti-Ub antibody. The U2OS cells were treated with DMSO or a combination of 10 µM RO3306 and 10 µM CDK2i-III for 4 h before IP. e Ser89/Ser251 phosphorylation promotes ubiquitination of RECQL4. HA-tagged ubiquitin and 3xFLAG-tagged WT RECQL4 or RQ4-2A were co-transfected to HEK293T cells, and ubiquitinated proteins were pulled down by HA-IP. Ubiquitinated RECQL4 proteins in the IP product were detected by Western blotting with anti-FLAG antibody. f RQ4-2A showed lower affinity with DDB1 in HEK293T cells. 3xFLAG-tagged WT RECQL4, RQ4-2A, and RQ4-2D were pulled down from HEK293T cells and DDB1 protein was probed by Western blotting analysis. g Knockdown of DDB1 decreases recruitment of RECQL4 to laser-induced DSBs. DDB1 levels in control and DDB1-depleted U2OS cells were determined by Western blotting. DSBs were generated by micro-point laser. Real-time recruitment of GFP-RECQL4 was observed and quantified. Relative intensity of GFP-RECQL4 is shown as mean ± s.e.m. with p-value by Student’s t-test. The number of cells analyzed : for control cells, n = 17; shDDB1, n = 24. Scale bar, 5 µm. h Depletion of CUL4A in U2OS cells caused failure of RECQL4 recruitment at chromatin after IR stress. Subcellular fractionations of control or CUL4A siRNA-treated U2OS cells were prepared and indicated proteins were examined with Western blotting in these fractions

false

• WB

CiteAb

Western blot - Anti-DDB1 antibody [EPR6089] (AB109027)

Western Blotting using Anti-DDB1 antibody [EPR6089], ab109027. Publication image from Lu, H. et al., 2017, Nat Commun, 29229926. Legend direct from paper.

DDB1-CUL4A E3 ubiquitin ligase stimulates RECQL4 recruitment to DSBs. a Co-IP analysis for the interaction of RECQL4 with DDB1 and CUL4A in HEK293T cells. DDB1 and CUL4A were pulled down with 3xFLAG-tagged RECQL4 from HEK293T cells. b N-terminal domain of RECQL4 interacts with DDB1 in HEK293T cells. Upper panel shows schematic diagram of 3xFLAG-tagged RECQL4 and the truncated fragments. N-terminal domain (RQ4-NT), helicase domain (RQ4-HE), and C-terminal domain (RQ4-CT) were tagged with both 3xFLAG and SV40 nuclear-location sequence (NLS). c Knockdown of either DDB1 or CUL4A inhibits ubiquitination of RECQL4 in U2OS cells. Endogenous RECQL4 was purified with anti-RECQL4 antibody from denatured U2OS cell lysates and the ubiquitination was then detected with anti-Ub antibody. d Inhibition of CDK1 and CDK2 reduced the ubiquitination of RECQL4 in U2OS cells. RECQL4 was immunoprecipitated with anti-RECQL4 antibody from U2OS cells, and its ubiquitination was examined with anti-Ub antibody. The U2OS cells were treated with DMSO or a combination of 10 µM RO3306 and 10 µM CDK2i-III for 4 h before IP. e Ser89/Ser251 phosphorylation promotes ubiquitination of RECQL4. HA-tagged ubiquitin and 3xFLAG-tagged WT RECQL4 or RQ4-2A were co-transfected to HEK293T cells, and ubiquitinated proteins were pulled down by HA-IP. Ubiquitinated RECQL4 proteins in the IP product were detected by Western blotting with anti-FLAG antibody. f RQ4-2A showed lower affinity with DDB1 in HEK293T cells. 3xFLAG-tagged WT RECQL4, RQ4-2A, and RQ4-2D were pulled down from HEK293T cells and DDB1 protein was probed by Western blotting analysis. g Knockdown of DDB1 decreases recruitment of RECQL4 to laser-induced DSBs. DDB1 levels in control and DDB1-depleted U2OS cells were determined by Western blotting. DSBs were generated by micro-point laser. Real-time recruitment of GFP-RECQL4 was observed and quantified. Relative intensity of GFP-RECQL4 is shown as mean ± s.e.m. with p-value by Student’s t-test. The number of cells analyzed : for control cells, n = 17; shDDB1, n = 24. Scale bar, 5 µm. h Depletion of CUL4A in U2OS cells caused failure of RECQL4 recruitment at chromatin after IR stress. Subcellular fractionations of control or CUL4A siRNA-treated U2OS cells were prepared and indicated proteins were examined with Western blotting in these fractions

false

• WB

CiteAb

Western blot - Anti-DDB1 antibody [EPR6089] (AB109027)

Western Blotting using Anti-DDB1 antibody [EPR6089], ab109027. Publication image from Lu, H. et al., 2017, Nat Commun, 29229926. Legend direct from paper.

DDB1-CUL4A E3 ubiquitin ligase stimulates RECQL4 recruitment to DSBs. a Co-IP analysis for the interaction of RECQL4 with DDB1 and CUL4A in HEK293T cells. DDB1 and CUL4A were pulled down with 3xFLAG-tagged RECQL4 from HEK293T cells. b N-terminal domain of RECQL4 interacts with DDB1 in HEK293T cells. Upper panel shows schematic diagram of 3xFLAG-tagged RECQL4 and the truncated fragments. N-terminal domain (RQ4-NT), helicase domain (RQ4-HE), and C-terminal domain (RQ4-CT) were tagged with both 3xFLAG and SV40 nuclear-location sequence (NLS). c Knockdown of either DDB1 or CUL4A inhibits ubiquitination of RECQL4 in U2OS cells. Endogenous RECQL4 was purified with anti-RECQL4 antibody from denatured U2OS cell lysates and the ubiquitination was then detected with anti-Ub antibody. d Inhibition of CDK1 and CDK2 reduced the ubiquitination of RECQL4 in U2OS cells. RECQL4 was immunoprecipitated with anti-RECQL4 antibody from U2OS cells, and its ubiquitination was examined with anti-Ub antibody. The U2OS cells were treated with DMSO or a combination of 10 µM RO3306 and 10 µM CDK2i-III for 4 h before IP. e Ser89/Ser251 phosphorylation promotes ubiquitination of RECQL4. HA-tagged ubiquitin and 3xFLAG-tagged WT RECQL4 or RQ4-2A were co-transfected to HEK293T cells, and ubiquitinated proteins were pulled down by HA-IP. Ubiquitinated RECQL4 proteins in the IP product were detected by Western blotting with anti-FLAG antibody. f RQ4-2A showed lower affinity with DDB1 in HEK293T cells. 3xFLAG-tagged WT RECQL4, RQ4-2A, and RQ4-2D were pulled down from HEK293T cells and DDB1 protein was probed by Western blotting analysis. g Knockdown of DDB1 decreases recruitment of RECQL4 to laser-induced DSBs. DDB1 levels in control and DDB1-depleted U2OS cells were determined by Western blotting. DSBs were generated by micro-point laser. Real-time recruitment of GFP-RECQL4 was observed and quantified. Relative intensity of GFP-RECQL4 is shown as mean ± s.e.m. with p-value by Student’s t-test. The number of cells analyzed : for control cells, n = 17; shDDB1, n = 24. Scale bar, 5 µm. h Depletion of CUL4A in U2OS cells caused failure of RECQL4 recruitment at chromatin after IR stress. Subcellular fractionations of control or CUL4A siRNA-treated U2OS cells were prepared and indicated proteins were examined with Western blotting in these fractions

false

• WB

CiteAb

Western blot - Anti-DDB1 antibody [EPR6089] (AB109027)

Western Blotting using Anti-DDB1 antibody [EPR6089], ab109027. Publication image from Lu, H. et al., 2017, Nat Commun, 29229926. Legend direct from paper.

DDB1-CUL4A E3 ubiquitin ligase stimulates RECQL4 recruitment to DSBs. a Co-IP analysis for the interaction of RECQL4 with DDB1 and CUL4A in HEK293T cells. DDB1 and CUL4A were pulled down with 3xFLAG-tagged RECQL4 from HEK293T cells. b N-terminal domain of RECQL4 interacts with DDB1 in HEK293T cells. Upper panel shows schematic diagram of 3xFLAG-tagged RECQL4 and the truncated fragments. N-terminal domain (RQ4-NT), helicase domain (RQ4-HE), and C-terminal domain (RQ4-CT) were tagged with both 3xFLAG and SV40 nuclear-location sequence (NLS). c Knockdown of either DDB1 or CUL4A inhibits ubiquitination of RECQL4 in U2OS cells. Endogenous RECQL4 was purified with anti-RECQL4 antibody from denatured U2OS cell lysates and the ubiquitination was then detected with anti-Ub antibody. d Inhibition of CDK1 and CDK2 reduced the ubiquitination of RECQL4 in U2OS cells. RECQL4 was immunoprecipitated with anti-RECQL4 antibody from U2OS cells, and its ubiquitination was examined with anti-Ub antibody. The U2OS cells were treated with DMSO or a combination of 10 µM RO3306 and 10 µM CDK2i-III for 4 h before IP. e Ser89/Ser251 phosphorylation promotes ubiquitination of RECQL4. HA-tagged ubiquitin and 3xFLAG-tagged WT RECQL4 or RQ4-2A were co-transfected to HEK293T cells, and ubiquitinated proteins were pulled down by HA-IP. Ubiquitinated RECQL4 proteins in the IP product were detected by Western blotting with anti-FLAG antibody. f RQ4-2A showed lower affinity with DDB1 in HEK293T cells. 3xFLAG-tagged WT RECQL4, RQ4-2A, and RQ4-2D were pulled down from HEK293T cells and DDB1 protein was probed by Western blotting analysis. g Knockdown of DDB1 decreases recruitment of RECQL4 to laser-induced DSBs. DDB1 levels in control and DDB1-depleted U2OS cells were determined by Western blotting. DSBs were generated by micro-point laser. Real-time recruitment of GFP-RECQL4 was observed and quantified. Relative intensity of GFP-RECQL4 is shown as mean ± s.e.m. with p-value by Student’s t-test. The number of cells analyzed : for control cells, n = 17; shDDB1, n = 24. Scale bar, 5 µm. h Depletion of CUL4A in U2OS cells caused failure of RECQL4 recruitment at chromatin after IR stress. Subcellular fractionations of control or CUL4A siRNA-treated U2OS cells were prepared and indicated proteins were examined with Western blotting in these fractions

false

• WB

CiteAb

Western blot - Anti-DDB1 antibody [EPR6089] (AB109027)

Western Blotting using Anti-DDB1 antibody [EPR6089], ab109027. Publication image from Lu, H. et al., 2017, Nat Commun, 29229926. Legend direct from paper.

DDB1-CUL4A E3 ubiquitin ligase stimulates RECQL4 recruitment to DSBs. a Co-IP analysis for the interaction of RECQL4 with DDB1 and CUL4A in HEK293T cells. DDB1 and CUL4A were pulled down with 3xFLAG-tagged RECQL4 from HEK293T cells. b N-terminal domain of RECQL4 interacts with DDB1 in HEK293T cells. Upper panel shows schematic diagram of 3xFLAG-tagged RECQL4 and the truncated fragments. N-terminal domain (RQ4-NT), helicase domain (RQ4-HE), and C-terminal domain (RQ4-CT) were tagged with both 3xFLAG and SV40 nuclear-location sequence (NLS). c Knockdown of either DDB1 or CUL4A inhibits ubiquitination of RECQL4 in U2OS cells. Endogenous RECQL4 was purified with anti-RECQL4 antibody from denatured U2OS cell lysates and the ubiquitination was then detected with anti-Ub antibody. d Inhibition of CDK1 and CDK2 reduced the ubiquitination of RECQL4 in U2OS cells. RECQL4 was immunoprecipitated with anti-RECQL4 antibody from U2OS cells, and its ubiquitination was examined with anti-Ub antibody. The U2OS cells were treated with DMSO or a combination of 10 µM RO3306 and 10 µM CDK2i-III for 4 h before IP. e Ser89/Ser251 phosphorylation promotes ubiquitination of RECQL4. HA-tagged ubiquitin and 3xFLAG-tagged WT RECQL4 or RQ4-2A were co-transfected to HEK293T cells, and ubiquitinated proteins were pulled down by HA-IP. Ubiquitinated RECQL4 proteins in the IP product were detected by Western blotting with anti-FLAG antibody. f RQ4-2A showed lower affinity with DDB1 in HEK293T cells. 3xFLAG-tagged WT RECQL4, RQ4-2A, and RQ4-2D were pulled down from HEK293T cells and DDB1 protein was probed by Western blotting analysis. g Knockdown of DDB1 decreases recruitment of RECQL4 to laser-induced DSBs. DDB1 levels in control and DDB1-depleted U2OS cells were determined by Western blotting. DSBs were generated by micro-point laser. Real-time recruitment of GFP-RECQL4 was observed and quantified. Relative intensity of GFP-RECQL4 is shown as mean ± s.e.m. with p-value by Student’s t-test. The number of cells analyzed : for control cells, n = 17; shDDB1, n = 24. Scale bar, 5 µm. h Depletion of CUL4A in U2OS cells caused failure of RECQL4 recruitment at chromatin after IR stress. Subcellular fractionations of control or CUL4A siRNA-treated U2OS cells were prepared and indicated proteins were examined with Western blotting in these fractions

false