Anti-Cullin 4A/CUL-4A antibody

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cullin 4A/CUL-4A antibody (AB72548)

IHC image of ab72548 staining in human normal cervix formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with EDTA (pH9, epitope retrieval solution 2) for 20 mins. The section was then incubated with ab72548, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cullin 4A/CUL-4A antibody (AB72548)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma (left) and mouse squamous cell carcinoma (right) tissues labelling Cullin 4A/CUL-4A with ab72548 at 1/1000 (1μg/ml). Detection : DAB.

• WB

Unknown

Western blot - Anti-Cullin 4A/CUL-4A antibody (AB72548)

All lanes:

Western blot - Anti-Cullin 4A/CUL-4A antibody (ab72548) at 0.1 µg/mL

Lane 1:

HeLa whole cell lysate at 50 µg

Lane 2:

HeLa whole cell lysate at 15 µg

Lane 3:

HeLa whole cell lysate at 5 µg

Lane 4:

293T whole cell lysate at 50 µg

Lane 5:

NIH3T3 whole cell lysate at 50 µg

Predicted band size: 88 kDa

Observed band size: 117 kDa,88 kDa

false

• WB

CiteAb

Western blot - Anti-Cullin 4A/CUL-4A antibody (AB72548)

Western Blotting using Anti-Cullin 4A/CUL-4A antibody, ab72548. 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-Cullin 4A/CUL-4A antibody (AB72548)

Western Blotting using Anti-Cullin 4A/CUL-4A antibody, ab72548. 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-Cullin 4A/CUL-4A antibody (AB72548)

Western Blotting using Anti-Cullin 4A/CUL-4A antibody, ab72548. 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