Phospho-GSK3 (alpha + beta) (Y216 + Y279) Recombinant Rabbit Monoclonal Antibody [SY26-05]

Overview

Product Name

Phospho-GSK3 (alpha + beta) (Y216 + Y279) Recombinant Rabbit Monoclonal Antibody [SY26-05] - BSA and Azide free

Antibody Type

Recombinant Rabbit monoclonal Antibody

Immunogen

Synthetic phospho-peptide corresponding to residues surrounding Tyr216 and 279 of human GSK3(alpha+beta).

Species Reactivity

Human, Mouse, Rat

Validated Applications

WB, IF-Cell, IHC-P, IP, IF-Tissue, FC

Molecular Weight

Predicted band size: 51 kDa

Positive Control

293T cell lysate, SH-SY5Y cell lysate, HeLa cell lysate, HepG2 cell lysate, MCF7 cell lysate, NIH/3T3 cell lysate, PC-12 cell lysate, mouse brain tissue lysate, rat brain tissue lysate, mouse cerebellum tissue lysates, A431, Hela, MCF-7, rat brain tissue, rat hippocampus tissue, mouse brain tissue, human thyroid carcinoma tissue.

Conjugation

unconjugated

Clone Number

SY26-05

Product Features

Form

Liquid

Concentration

1mg/ml

Storage Instructions

Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles.

Storage Buffer

1*PBS (pH7.4).

Isotype

IgG

Purification Method

Protein A affinity purified.

Application Dilution

WB
1:500-1:1,000

IF-Cell
1:100-1:500

IHC-P
1:200-1:800

IP
Use at an assay dependent concentration.

IF-Tissue
1:200

FC
1:1,000

Target

Function

Glycogen synthase kinase-3α (GSK-3α) and GSK-3β are highly similar isoforms of serine/ threonine kinases that regulate metabolic enzymes and transcription factors, which are responsible for coordinating processes such as glycogen synthesis and cell adhesion. GSK-3β activity is also required for nuclear activity of Rel dimers, which mediate an anti-apoptotic response to TNFα in mice. GSK-3 catalytic kinase activity is controlled through differential phosphorylation of serine/threonine residues, which have an inhibitory effect, and tyrosine residues, which have an activating effect. Growth factor stimulation of mammalian cells expressing GSK-3α and GSK-3β induces phosphorylation of Ser 21 and Ser 9, respectively, through a phosphatidylinositol 3-kinase (PI 3-K)-protein kinase B (PKB)-dependent pathway, thereby enhancing proliferative signals. Additionally, GSK-3 physically associates with cAMP-dependent protein kinase A (PKA), which phosphorylates Ser 21 of GSK-3α or Ser 9 of GSK-3β and inactivates both forms. GSK-3α/β is positively regulated by phosphorylation on Tyr 279 and Tyr 216, respectively. Activated GSK-3α/β participates in energy metabolism, neuronal cell development, and body pattern formation. Tyrosine dephosphorylation of GSK-3 is involved in its extracellular signal-dependent inactivation.

Background References

1. Xu R et al. The protease Omi regulates mitochondrial biogenesis through the GSK3 /PGC-1a pathway. Cell Death Dis 5:e1373 (2014).

2. Weigand S et al. Global quantitative phosphoproteome analysis of human tumor xenografts treated with a CD44 antagonist. Cancer Res 72:4329-39 (2012).

Sequence Similarity

Belongs to the protein kinase superfamily. CMGC Ser/Thr protein kinase family. GSK-3 subfamily.

Post-translational Modification

Phosphorylated by AKT1 at Ser-21: upon insulin-mediated signaling, the activated PKB/AKT1 protein kinase phosphorylates and desactivates GSK3A, resulting in the dephosphorylation and activation of GYS1. Activated by phosphorylation at Tyr-279.

Subcellular Location

Cytoplasm, Nucleus, Cell membrane.

UNIPROT

SwissProt: P49840 Human

SwissProt: P49841 Human

SwissProt: Q2NL51 Mouse

SwissProt: Q9WV60 Mouse

SwissProt: P18265 Rat

SwissProt: P18266 Rat

Synonyms

Factor A antibody

Glycogen synthase kinase 3 alpha antibody

Glycogen synthase kinase 3 beta antibody

GSK3 alpha antibody

GSK3 beta antibody

GSK3B antibody

Images

☑ Cell treatment (CT)

Western blot analysis of Phospho-GSK3 (alpha + beta) (Y216 + Y279) on different lysates with Rabbit anti-Phospho-GSK3 (alpha + beta) (Y216 + Y279) antibody (HA750127) at 1/5,000 dilution.

Lane 1: 293T cell lysate
Lane 2: SH-SY5Y cell lysate
Lane 3: HeLa cell lysate
Lane 4: HepG2 cell lysate
Lane 5: MCF7 cell lysate
Lane 6: NIH/3T3 cell lysate
Lane 7: PC-12 cell lysate
Lane 8: Mouse brain tissue lysate
Lane 9: Rat brain tissue lysate
Lane 10: 293T treated with λpp for 1 hour cell lysate
Lane 11: SH-SY5Y treated with λpp for 1 hour cell lysate

Lysates/proteins at 15 µg/Lane.

Predicted band size: 47/51 kDa
Observed band size: 47/51 kDa

Exposure time: 5 minutes;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA750127) at 1/5,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:50,000 dilution was used for 1 hour at room temperature.
ICC staining of Phospho-GSK3 (alpha + beta) (Y216 + Y279) in A431 cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 10% negative goat serum for 15 minutes at room temperature. Cells were probed with the primary antibody (HA750127, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 conjugate-Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).
ICC staining of Phospho-GSK3 (alpha + beta) (Y216 + Y279) in Hela cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 10% negative goat serum for 15 minutes at room temperature. Cells were probed with the primary antibody (HA750127, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 conjugate-Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).
ICC staining of Phospho-GSK3 (alpha + beta) (Y216 + Y279) in MCF-7 cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 10% negative goat serum for 15 minutes at room temperature. Cells were probed with the primary antibody (HA750127, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 conjugate-Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).
☑ Cell treatment (CT)

Western blot analysis of Phospho-GSK3(alpha+beta)(Y216+Y279) on mouse cerebellum tissue lysates.

Lane 1: mouse cerebellum tissue, whole cell lysate, 20ug/lane
Lane 2: mouse cerebellum tissue treated with 2.8ug/ul lambda-PP for 30 minutes, whole cell lysates, 20ug/lane

All lanes :
Anti-Phospho-GSK3(alpha+beta)(Y216+Y279) antibody (HA750127) at 1:500 dilution. Anti-GAPDH antibody (ET1601-4) at 1:10,000 dilution. Goat Anti-Rabbit IgG H&L (HRP) (HA1001) at 1/200,000 dilution.

Predicted band size: 47/51 kDa
Observed band size: 47/51 kDa

Blocking and diluting buffer: 5% BSA.

Exposure time: 2 minutes
Immunohistochemical analysis of paraffin-embedded rat brain tissue with Rabbit anti-Phospho-GSK3 (alpha + beta) (Y216 + Y279) antibody (HA750127) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (HA750127) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
Immunohistochemical analysis of paraffin-embedded rat hippocampus tissue with Rabbit anti-Phospho-GSK3 (alpha + beta) (Y216 + Y279) antibody (HA750127) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (HA750127) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
Immunohistochemical analysis of paraffin-embedded mouse brain tissue with Rabbit anti-Phospho-GSK3 (alpha + beta) (Y216 + Y279) antibody (HA750127) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (HA750127) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
Immunohistochemical analysis of paraffin-embedded human thyroid carcinoma tissue with Rabbit anti-Phospho-GSK3 (alpha + beta) (Y216 + Y279) antibody (HA750127) at 1/800 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (HA750127) at 1/800 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
Flow cytometric analysis of HeLa cells labeling Phospho-GSK3 (alpha + beta) (Y216 + Y279).

Cells were fixed and permeabilized. Then stained with the primary antibody (HA750127, 1μg/mL) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
Flow cytometric analysis of NIH/3T3 cells labeling Phospho-GSK3 (alpha + beta) (Y216 + Y279).

Cells were fixed and permeabilized. Then stained with the primary antibody (HA750127, 1μg/mL) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

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