Anti-CDK1 antibody [EPR165]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CDK1 antibody [EPR165] (AB133327)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labelling CDK1 with ab133327 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) antibody at 1/1000 dilution (Green).

Anti-alpha Tubulin mouse monoclonal antibody (ab7291)  was used to counterstain tubulin at 1/1000 dilution (Magenta).

Confocal image showing nucleus and cytoplasm staining in HeLa cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Secondary antibody only control : Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) at 1/1000 dilution.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-CDK1 antibody [EPR165] (AB133327)

Intracellular Flow Cytometry analysis of Jurkat (human acute T cell leukemia) cells labeling CDK1 with purified ab133327 at 1/130 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) was used as the unlabeled control.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CDK1 antibody [EPR165] (AB133327)

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling CDK1 with ab133327 at 1/500 dilution (0.6 μg/ml). Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889, an anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1/200 (2.5 μg/ml). ab150077, a Goat anti-rabbit IgG (Alexa Fluor^® 488) was used as the secondary antibody at 1/1000 dilution (2 μg/ml). DAPI nuclear counterstain. Confocal image showing strong nuclear and weakly cytoplasmic staining on HeLa cell line.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CDK1 antibody [EPR165] (AB133327)

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labelling CDK1 with ab133327 at 1/250 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• IP

Lab

Immunoprecipitation - Anti-CDK1 antibody [EPR165] (AB133327)

CDK1 was immunoprecipitated from 0.35 mg HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10 μg with ab133327 at 1/20 dilution (2μg) . VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10 μg

Lane 2 : ab133327 IP in HeLa whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab133327 in HeLa whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-CDK1 antibody [EPR165] (ab133327)

Predicted band size: 34 kDa

Observed band size: 34 kDa

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• WB

Unknown

Western blot - Anti-CDK1 antibody [EPR165] (AB133327)

All lanes:

Western blot - Anti-CDK1 antibody [EPR165] (ab133327) at 1/10000 dilution

Lane 1:

HeLa cell lysate at 10 µg

Lane 2:

Saos-2 cell lysate at 10 µg

Lane 3:

Namalwa cell lysate at 10 µg

Lane 4:

Jurkat cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 34 kDa

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• WB

CiteAb

Western blot - Anti-CDK1 antibody [EPR165] (AB133327)

Western Blotting using Anti-CDK1 antibody [EPR165], ab133327. Publication image from Liu, J. et al., 2020, Nat Commun, 32198345. Legend direct from paper.

AGPG is required for cell proliferation and metabolism remodeling.a qPCR and electrophoresis detection of AGPG expression in KYSE30 and KYSE150 cells. Ctrl, control. b Cell proliferation was assessed by MTS assays (OD 490 nm). c, d Colony formation assays and statistical analysis of ESCC cells transduced with shAGPG #1 or #2 or shCtrl. e The cell cycle was analyzed by flow cytometry analysis. f Statistical analysis of KYSE150 cells (%) in each cell cycle phase. g CDK1 and p27 expression levels were detected by western blotting in cells transfected with shAGPG #1 or #2 or shCtrl. h The ECAR was measured in cells transfected with shAGPG #1 or #2 or shCtrl using an XF Extracellular Flux Analyzer. i Statistical analysis of the effects of AGPG knockdown on glycolytic activity. j Flowchart of the experiments for identifying the role of AGPG in glucose metabolism. k–m13C-Labeled metabolic intermediates of glycolysis were decreased after AGPG knockdown. Data in b, d, f, i, k–m are representative of three independent experiments and presented as mean±S.D., n = 3 biologically independent samples, the P value in b, d, f, i was determined by one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test, no adjustments were made for multiple comparisons. The P value in k–m was determined by a two-tailed unpaired Student’s t test.

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• WB

CiteAb

Western blot - Anti-CDK1 antibody [EPR165] (AB133327)

Western Blotting using Anti-CDK1 antibody [EPR165], ab133327. Publication image from Liu, J. et al., 2020, Nat Commun, 32198345. Legend direct from paper.

AGPG directly associates with PFKFB3.a, b PFKFB3 in cell lysates a or purified His-tagged recombinant PFKFB3 b was pulled down by biotin-labeled AGPG but not by AGPG antisense RNA. S, sense. AS, antisense. c RIP assays indicated that AGPG precipitated with PFKFB3 in whole-cell lysates. The RNA levels of AGPG and β-actin were measured by qPCR analysis. dAGPG-binding proteins were detected by MTRAP and western blotting analysis. PFKFB3 bound to AGPG was captured by anti-FLAG antibody affinity agarose beads; IP complexes were separated and identified by specific antibodies. e Immunofluorescence analysis showed that AGPG and PFKFB3 colocalized not only in the nucleus but also in the cytoplasm. Scale bar : 5 µm. f qPCR detection of AGPG expression and western blotting detection of PFKFB3 expression in human ESCC cells. PFKFB3 expression was positively correlated with AGPG expression. (Pearson’s correlation analysis, n = 10). g In vitro-synthesized FL and truncation mutants of AGPG were incubated with protein lysates from KYSE150 and KYSE30 cells or with purified His-tagged recombinant PFKFB3. RNA pull-down and western blotting assays were then performed. h CLIP-qPCR showed that the T5 fragment of AGPG was the region responsible for PFKFB3 binding. i RNA pull-down assays showed that AGPG δT5 could not interact with PFKFB3. jAGPG CRISPR KO cell lines were generated using the CRISPR/Cas9 genome-editing system. Overexpression of AGPG FL, but not of AGPG δT5, was sufficient to reverse the decreased ECAR and cell proliferation caused by AGPG CRISPR KO. k Western blotting showed that CDK1 downregulation and p27 upregulation by AGPG CRISPR KO were abolished by AGPG FL but not by AGPG δT5. l HomeR was used to perform the motif analysis on the binding peaks obtained by the Piranha and CIMS analyses. Both methods suggested that CCAGCCA might be responsible for PFKFB3 binding. Data in c, f, h, j are representative of three independent experiments and presented as mean±S.D., n = 3 biologically independent samples, the P value was determined by a two-tailed unpaired Student’s t test.

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• WB

CiteAb

Western blot - Anti-CDK1 antibody [EPR165] (AB133327)

Western Blotting using Anti-CDK1 antibody [EPR165], ab133327. Publication image from Lu, H. et al., 2017, Nat Commun, 29229926. Legend direct from paper.

CDK1/2-dependent phosphorylation promotes RECQL4 participation in repair of DSBs. a–c In vitro phosphorylation of RECQL4 by CDK1/Cyclin A (a), CDK2/Cyclin A (b), and CDK2/Cyclin E (c). 3xFLAG-tagged RECQL4 WT and RQ4-2A mutant were purified from HEK293T cells, and pre-treated with λPP. After washing off λPP, the RECQL4 proteins were incubated with CDK1/2 and their cyclin partners. Phosphorylation of RECQL4 SP sites were analyzed by Western blotting. d CDK1/2 inhibitors repress p-SP level of RECQL4. HEK293T cells expressing 3xFLAG-RECQL4 were pretreated with 10 µM RO3306 (CDK1i), 10 µM CDK2i-III (CDK2i) or combination of CDK1i and CDK2i for 4 h. Then, 3xFLAG-tagged RECQL4 were purified and SP sites’ phosphorylation was analyzed by Western blotting. e Ser89/Ser251 phosphorylation promotes recruitment of RECQL4 to laser-induced DSBs. GFP-tagged RECQL4 Wt, RQ4-2A, and RQ4-2D were expressed in U2OS cells, and the cells were stripped with micro-point laser after treatment with CDK1 and CDK2 inhibitors or DMSO. The relative intensities of these cells are presented as mean ± s.e.m. The number of cells were quantified, for RQ4Wt, n = 9; RQ4Wt with CDK1/2i, n = 16; RQ4-2A, n = 19; RQ4-2A with CDK1/2i, n = 17; RQ4-2D, n = 26. RQ4-2D with CDK1/2i, n = 19. Scale bar, 5 µm. f Inhibition of CDK1 and CDK2 attenuated recruitment of YFP-MRE11 to laser-induced DSBs. For DMSO, n = 27; for CDK1/2i, n = 49. g Inhibition of CDK1 and CDK2 represses RECQL4’s interaction with MRE11. The U2OS cells pretreated with CDK1 and CDK2 inhibitors or DMSO were irradiated for 10 Gy and processed for IP with anti-MRE11 antibody 5 min after irradiation. h Ser89/Ser251 phosphorylation promotes the interaction of RECQL4 with MRE11. GFP-tagged RECQL4, RQ4-2A, and RQ4-2D were immunoprecipitated from HEK293T cells after a 10 Gy IR treatment. MRE11 and Ku70 were measured in the IP products

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