Anti-MCM2 antibody

• IP

Supplier Data

Immunoprecipitation - Anti-MCM2 antibody (AB4461)

MCM2 was immunoprecipitated from 1 mg per reaction of HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate with ab4461 at 3 μg per reaction. Western blot was performed from the immunoprecipitate using ab4461 at 1 μg/ml.

Lane 1 : Phospho MCM2 (S108) antibody IP in HEK-293T lysate.
Lane 2 : ab4461 IP in HEK-293T lysate.
Lane 3 : Control IgG instead of ab4461 in HEK-293T lysate.

NETN lysis buffer.
Exposure time : XXXX.

All lanes:

Immunoprecipitation - Anti-MCM2 antibody (ab4461)

Predicted band size: 102 kDa

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• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MCM2 antibody (AB4461)

ab4461 staining MCM2 in Human placenta.
Left panel : with primary antibody at 4 ug/ml. Right panel : isotype control.
Sections were stained using an automated system (DAKO Autostainer Plus), at room temperature : sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffers EDTA pH 9.0. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MCM2 antibody (AB4461)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human small cell lung cancer tissue labeling MCM2 with ab4461 at 1/1000 (1μg/ml). Detection : DAB.

• WB

Supplier Data

Western blot - Anti-MCM2 antibody (AB4461)

All lanes:

Western blot - Anti-MCM2 antibody (ab4461) at 0.1 µg/mL

Lane 1:

HeLa (Human epithelial adenocarcinoma cell line) whole cell lysate at 50 µg

Lane 2:

HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate at 50 µg

Lane 3:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 50 µg

Lane 4:

TCMK-1 (Mouse kidney epithelial cell line) whole cell lysate at 50 µg

Lane 5:

NIH/3T3 (Mouse embryo fibroblast cell line) whole cell lysate at 50 µg

Predicted band size: 102 kDa

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• WB

CiteAb

Western blot - Anti-MCM2 antibody (AB4461)

Western Blotting using Anti-MCM2 antibody, ab4461. Publication image from Storchova, Z. et al., 2016, Nat Commun, 26876972. Legend direct from paper.

The abundance of replication factors is decreased in response to extra chromosomes.(a) Unsupervised hierarchical clustering of the protein abundance fold changes (calculated log2 aneuploid-to-diploid ratio) of factors assigned to KEGG (Kyoto Encyclopedia of Genes and Genomes)-defined term replication; manually curated. HCT116 5/4 and HCT116 3/3 marked in the figure in bold are cell lines previously constructed by Minoru Koi; these cell lines were used only for the global proteome analysis. See Methods for more details. (b) Immunoblotting of subunits of the replicative helicase MCM2-7 in whole-cell extracts from trisomic cell lines and their respective controls. (c,d) Levels of chromatin-bound replication proteins in the parental cell lines and the trisomic and tetrasomic cell lines. Note that MCM2, 3 and 7 are downregulated in all cell lines with extra chromosomes except for RPE1 5/3 12/3 c3. Ponceau staining was used as loading control. (e,f) Levels of chromatin-bound subunits of the MCM2-7 helicase in asynchronous and synchronized cells in HCT116 5/3 and RPE1 21/3, and respective controls.

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• WB

CiteAb

Western blot - Anti-MCM2 antibody (AB4461)

Western Blotting using Anti-MCM2 antibody, ab4461. Publication image from Raynaud, P. et al., 2017, Nat Commun, 28785014. Legend direct from paper.

Ensa KD increases S-phase length by decreasing fork number without affecting origin licensing. a Diagram depicting IdU (red)–CldU (green) double pulse analysis by DNA combing. b Representative image of a triple-stained DNA fibre : IdU (red)–CldU (green)–DNA (blue). c Fork velocity (Kb/min) and d global instant fork density (GIFD) values (fork per Mb) are represented. Mean ± standard deviation and p-value obtained by using two-tailed unpaired Student’s t-tests, are represented. Data pooled from four biological replicates. e Diagram representing the experiment in which HeLa cells were blocked in G1–S phase by thymidine for 24 h and subsequently released with fresh medium containing nocodazole (50 ng/ml) for a supplementary period of 16 h before shakeoff. Four hours later, released cells were transfected with siSC or siEnsa, and after 1 h, supplemented with fresh medium containing thymidine for an additional 18 h incubation. Cells were then released and recovered at the indicated times for FACS analysis. f FACS profiles of SC (red) and Ensa (blue) siRNA-treated cells at different time points after release are shown. g HeLa and U2OS cells were transfected with scramble (siSC) or Ensa (siEnsa) siRNAs and 48 h later processed for chromatin isolation as reported in Methods section. Cytosolic (S2) and nuclear (S3) soluble fractions as well as chromatin fraction (P3) were then used for western blot analysis to determine Treslin and MCM 2, 3 and 4 levels. h As for g, except that MCM 5 and 7, histone H3 and β-tubulin levels were checked. IOD, inter-origin distances; Ori, replication origin

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• WB

CiteAb

Western blot - Anti-MCM2 antibody (AB4461)

Western Blotting using Anti-MCM2 antibody, ab4461. Publication image from Storchova, Z. et al., 2016, Nat Commun, 26876972. Legend direct from paper.

The accumulation of DNA damage is sensitive to abundance changes of MCM2-7 subunits.(a) Levels of total and chromatin-bound MCM2 and MCM7 in parental HCT116 upon siRNA-mediated depletion of MCM2. (b) Accumulation of 53BP1 and (c) anaphase bridges in HCT116 upon depletion of MCM2 with and without replication stress. All plots show mean±s.e.m. of three independent experiments, at least 500 cyclin A-negative cells or 50 anaphases were scored in each experiment. (d) Survival rates of HCT116, RPE1 and their trisomic and tetrasomic derivatives upon overexpression of wild-type and mutant alleles of MCM2 (MCM-457A). (e) Survival rates of HCT116, RPE1 and the trisomic and tetrasomic derivatives upon overexpression of wild-type and mutant alleles of ORC1 (ORC1δBAH). (f) Survival rates of HCT116, RPE1 and the trisomic and tetrasomic derivatives upon overexpression of wild-type and mutant alleles of RPA1 (RPA1 L221P). Survival rates were normalized to the control (pcDNA transfected sample). All plots show mean+s.e.m. of three independent experiments; nonparametric t-test; *P<0.05, **P<0.01, ***P<0.001. (g) Levels of total and chromatin-bound MCM2 and MCM7 in HCT116 5/3 upon transient overexpression of MCM7. (h) Formation of 53BP1 foci and (i) accumulation of anaphase bridges in HCT116 and HCT116 5/4 upon transient overexpression of MCM7. One representative plot of three independent experiments is shown. Nonparametric t-test; *P<0.05, **P<0.01, ***P<0.001.

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