Anti-ATM (phospho S1981) antibody [10H11.E12]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATM (phospho S1981) antibody [10H11.E12] (AB36810)

ab36810 staining human colon tissue. Staining is localized to the nucleus.
Left panel : with primary antibody at 4 μg/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 antigen retrieval buffer citrate pH 6.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H₂O₂ in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with diaminobenzidine for 5 minutes. Slides were counterstained with hematoxylin 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.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-ATM (phospho S1981) antibody [10H11.E12] (AB36810)

Overlay histogram showing HeLa cells stained with ab36810 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab36810, 1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

• WB

Unknown

Western blot - Anti-ATM (phospho S1981) antibody [10H11.E12] (AB36810)

Lysate loading concentration : 40µg

All lanes:

Western blot - Anti-ATM (phospho S1981) antibody [10H11.E12] (ab36810) at 1/1000 dilution

Lane 1:

Control lane

Lane 2:

Irradiated Human fibroblasts (10 Gy gamma-irradiation)

Lane 3:

Molecular weight marker

Lane 4:

Peroxidated Human fibroblasts (300 µM hydrogen peroxide)

Lane 5:

Peroxidated Human fibroblasts (1 mM hydrogen peroxide)

Lane 6:

Peroxidated Human fibroblasts (10 mM hydrogen peroxide)

Predicted band size: 351 kDa

Observed band size: 370 kDa

true

• WB

Unknown

Western blot - Anti-ATM (phospho S1981) antibody [10H11.E12] (AB36810)

All lanes:

Western blot - Anti-ATM (phospho S1981) antibody [10H11.E12] (ab36810) at 10 µg/mL

Lane 1:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 20 µg

Lane 2:

Extract from patient with Ataxia-Telangiectasia whole cell lysate at 20 µg

Lane 3:

Irradiated HeLa Whole Cell Lysate at 20 µg

Secondary

All lanes:

Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Predicted band size: 351 kDa

Observed band size: 100 kDa,110 kDa,145 kDa,200 kDa,370 kDa

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Exposure time: 20min

• WB

CiteAb

Western blot - Anti-ATM (phospho S1981) antibody [10H11.E12] (AB36810)

Western Blotting using Anti-ATM (phospho S1981) antibody [10H11.E12], ab36810. Publication image from Gueven, N. et al., 2010, Mol Cancer, 20663147. Legend direct from paper.

p63 depletion attenuates ATM expression and ATM-dependent phosphorylation. (A) pSUPER-p63 RNAi attenuates ATM mRNA levels. HaCat cells were transfected with 1 µg pSUPER-CON or pSUPER-p63si vectors, and selected for geneticin resistance for 4 days. mRNA was extracted from surviving cells and real-time RT-PCR was used to quantitate changes in ATM mRNA expression. Data is represented as fold-change over empty vector control. (B) pSUPER-p63si inhibits ATM expression and p53 Serine-15 phosphorylation in HaCat cells. pSUPER-CON or pSUPER-p63si transfected HaCat cells were harvested after selection for 48 and 96 hours, and immunoblotted for the indicated proteins. (C) pSUPER-p63 RNAi inhibits HaCat cell proliferation. HaCaT cells were transfected with pSUPER-CON or pSUPER-p63si vectors, selected for geneticin resistance for 14 days, and surviving colonies were giemsa stained and counted. (D) siRNA-mediated p63 depletion attenuates ATM mRNA expression. HaCat cells untreated (NT) or treated with control siRNA or p63-targeted siRNA were harvested after 24 hrs or 48hrs and analysed by real-time RT-PCR for p63 and ATM mRNA expression. Data is normalised to β-actin and represented as fold-change over mRNA level in cells treated with control siRNA in particular time point. The data represent the average of three independent experiments + SD. The symbol asterisk denotes significant difference with p values < 0.05 determined by Mann-Whitney test, from the cells treated with control siRNA.

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• WB

CiteAb

Western blot - Anti-ATM (phospho S1981) antibody [10H11.E12] (AB36810)

Western Blotting using Anti-ATM (phospho S1981) antibody [10H11.E12], ab36810. Publication image from Gueven, N. et al., 2010, Mol Cancer, 20663147. Legend direct from paper.

The ATM pathway is active in HaCaT cells. (A) Damage-induced p53 Serine-15 is mediated by ATM. HaCat cells were treated with DMSO carrier, 10 µM KU-55933 (ATM inhibitor) or 1 µM NU7441 (DNA-PK inhibitor) for 2 hours, then with 0.5 µM doxorubicin, and cells were harvested after 2hrs, or cells were treated with 20Jm2 UV radiation and harvested after 6hrs. Lysates were blotted for phosphoSerine-15 p53, phosphoSerine-392 p53, total p53 protein and p63, as indicated. (B) ATM inhibition blocks constitutive p53 Serine-15 phosphorylation. Cells were treated with DMSO carrier, 10 µM KU-55933 (ATM inhibitor) or 1 µM NU7441 (DNA-PK inhibitor) for 24 hrs, and lysates were immunoblotted for phosphoSerine-15 p53 and total p53 protein. (C) ATM inhibition blocks constitutive ATM-dependent phosphorylation. Cells were treated with DMSO carrier or 10 µM KU-55933 for 24 hrs, and lysates were immunoblotted for : phosphoSerine-15 p53, phosphoSerine-392 p53, total p53 protein, phosphoThreonine-68 Chk2, total Chk2 protein, phosphoSerine-1891 ATM, and total ATM protein. (D) siRNA-mediated ATM depletion blocks p53 Serine-15 phosphorylation. HaCat cells were treated with control siRNA or siRNA to ATM for 24, 48 or 96hrs, and cell lysates were blotted for ATM, phosphoSerine-15 p53, and total p53 protein.

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