Anti-CDK1 + CDK2 + CDK3 + CDK5 (phospho Y15) antibody [EPR2233Y]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CDK1 + CDK2 + CDK3 + CDK5 (phospho Y15) antibody [EPR2233Y] (AB76146)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human transitional cell carcinoma of bladder tissue labelling Cdk1 + Cdk2 + Cdk3 + Cdk5 (phospho Y15) with purified ab76146 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer, pH 9. A prediluted HRP polymer-conjugated anti-rabbit IgG was used as the secondary antibody. Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

• IP

Lab

Immunoprecipitation - Anti-CDK1 + CDK2 + CDK3 + CDK5 (phospho Y15) antibody [EPR2233Y] (AB76146)

ab76146 (purified) at 1/100 immunoprecipitating Cdk1 + Cdk2 + Cdk3 + Cdk5 (phospho Y15) in HeLa cell lysates (Lane 1). Lane 2 - PBS. For western blotting, a HRP-conjugated anti-rabbit IgG, specific to the non-reduced form of IgG was used as the secondary antibody (1/1500).

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Immunoprecipitation - Anti-CDK1 + CDK2 + CDK3 + CDK5 (phospho Y15) antibody [EPR2233Y] (ab76146)

Observed band size: 33 kDa

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• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CDK1 + CDK2 + CDK3 + CDK5 (phospho Y15) antibody [EPR2233Y] (AB76146)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat colon tissue labelling Cdk1 + Cdk2 + Cdk3 + Cdk5 (phospho Y15) with purified ab76146 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer, pH 9. A prediluted HRP polymer-conjugated anti-rabbit IgG was used as the secondary antibody. Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

• WB

Lab

Western blot - Anti-CDK1 + CDK2 + CDK3 + CDK5 (phospho Y15) antibody [EPR2233Y] (AB76146)

Blocking/diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-CDK1 + CDK2 + CDK3 + CDK5 (phospho Y15) antibody [EPR2233Y] (ab76146) at 1/50000 dilution

Lane 1:

Untreated HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 15 µg

Lane 2:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate, treated with alkaline phosphatase for 1hour at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

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Exposure time: 10s

• WB

Lab

Western blot - Anti-CDK1 + CDK2 + CDK3 + CDK5 (phospho Y15) antibody [EPR2233Y] (AB76146)

ab76146 detects Cdk1, 2, 3, 5 phosphorylated on tyrosine 15.

All lanes:

Western blot - Anti-CDK1 + CDK2 + CDK3 + CDK5 (phospho Y15) antibody [EPR2233Y] (ab76146) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) transfected with GFP-tagged blank expression vector, whole cell lysate at 20 µg

Lane 2:

293T (human embryonic kidney epithelial cell) transfected with GFP-tagged CDK1 expression vector, whole cell lysate at 20 µg

Lane 3:

293T (human embryonic kidney epithelial cell) transfected with GFP-tagged CDK1 expression vector, whole cell lysate. Then the membrane was incubated with phosphatase at 20 µg

Lane 4:

293T (human embryonic kidney epithelial cell) transfected with GFP-tagged CDK2 expression vector, whole cell lysate at 20 µg

Lane 5:

293T (human embryonic kidney epithelial cell) transfected with GFP-tagged CDK2 expression vector, whole cell lysate. Then the membrane was incubated with phosphatase at 20 µg

Lane 6:

293T (human embryonic kidney epithelial cell) transfected with GFP-tagged CDK3 expression vector, whole cell lysate at 20 µg

Lane 7:

293T (human embryonic kidney epithelial cell) transfected with GFP-tagged CDK3 expression vector, whole cell lysate. Then the membrane was incubated with phosphatase at 20 µg

Lane 8:

293T (human embryonic kidney epithelial cell) transfected with GFP-tagged CDK5 expression vector, whole cell lysate at 20 µg

Lane 9:

293T (human embryonic kidney epithelial cell) transfected with GFP-tagged CDK5 expression vector, whole cell lysate. Then the membrane was incubated with phosphatase at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 34 kDa,65 kDa

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• Dot

Unknown

Dot Blot - Anti-CDK1 + CDK2 + CDK3 + CDK5 (phospho Y15) antibody [EPR2233Y] (AB76146)

Dot blot analysis of Cdk2 (pY15) peptide (Lane 1) and Cdk2 non-phospho peptide (Lane 2) labelling Cdk1 + Cdk2 + Cdk3 + Cdk5 (phospho Y15) with purified ab76146 at a dilution of 1/1000. ab97051 (Peroxidase conjugated goat anti-rabbit IgG (H+L)) was used as the secondary antibody at a dilution of 1/100000.

Blocking and dilution buffer : 5% NFDM/TBST.

Exposure time : 3 minutes.

• WB

CiteAb

Western blot - Anti-CDK1 + CDK2 + CDK3 + CDK5 (phospho Y15) antibody [EPR2233Y] (AB76146)

CDK1 + CDK2 + CDK3 + CDK5 (phospho Y15) western blot using anti-CDK1 + CDK2 + CDK3 + CDK5 (phospho Y15) antibody [EPR2233Y] ab76146. Publication image and figure legend from Liang, J., Cao, R., et al., 2016, Nat Commun, PubMed 27485204.

ab76146 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab76146 please see the product overview.

c-Src-phosphorylated Cdc25A Y59 dephosphorylates nuclear PKM2 pS37.Immunoprecipitation and immunoblot analyses were performed with the indicated antibodies. Data are representative of at least three independent experiments. (a) Flag-PKM2, which was phosphorylated at Ser 37 by EGF treatment (100 ng ml−1) for 3 h in U87/EGFR, was immunoprecipitated and eluted by Flag peptides. Immobilized and purified WT GST-Cdc25A or GST-Cdc25A Y59F was mixed with or without wild-type c-Src or kinase-dead c-Src K297M for an in vitro kinase assay. After the reaction, GST-Cdc25A was pulled down and incubated with eluted Flag-PKM2 in a phosphatase buffer for dephosphorylation reaction. (b,c) Endogenous Cdc25A-depleted U87/EGFR cells were reconstituted with the expression of rCdc25A WT or rCdc25A Y59F (b). These cells were treated with EGF (100 ng ml−1) for 6 h. Nuclear fractions of the cells were extracted (c). (d) U87/EGFR cells with depleted Cdc25A and reconstituted the expression of rCdc25A WT or rCdc25A Y59F were treated with or without EGF (100 ng ml−1) for 30 min. (e) An in vitro kinase assay was performed by mixing Cdc25A WT, Y59F mutant or C431S mutant with active c-Src. After the reaction, immunoblot analyses were performed with the reaction mixture (upper panel), or GST-Cdc25A WT and mutants were pulled down by glutathione agarose beads, and their activities toward FDP were examined using an Enzolyte FDP Protein Phosphatase Assay Kit (lower panel). Data represent the mean±s.d. of three independent experiments.

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