Anti-GSK3 beta (phospho Y216) + GSK3 alpha (phospho Y279) antibody [EPR933Y]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GSK3 beta (phospho Y216) + GSK3 alpha (phospho Y279) antibody [EPR933Y] (AB68476)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human thyroid carcinoma tissue labelling GSK3 (alpha + beta) with purified ab68476 at 1/2000. Heat mediated antigen retrieval was performed using Bond™ Epitope Retrieval Solution 2 (pH 9.0) for 20 minutes. ab209101, a Rabbit specific IHC polymer detection kit HRP/DAB was used. Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Positive staining on human thyroid carcinoma without alkaline phosphatase treatment (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B).

The section was incubated with ab68476 for 30 minutes at room temperature. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GSK3 beta (phospho Y216) + GSK3 alpha (phospho Y279) antibody [EPR933Y] (AB68476)

Human brain tissue stained with unpurified ab68476 at 1/100 dilution.

Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GSK3 beta (phospho Y216) + GSK3 alpha (phospho Y279) antibody [EPR933Y] (AB68476)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human ovarian carcinoma tissue labelling GSK3 (alpha + beta) with purified ab68476 at 1/2000. Heat mediated antigen retrieval was performed using Bond™ Epitope Retrieval Solution 2 (pH 9.0) for 20 minutes. ab209101, a Rabbit specific IHC polymer detection kit HRP/DAB was used. Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Positive staining on human ovarian carcinoma without alkaline phosphatase treatment (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B).

The section was incubated with ab68476 for 30 minutes at room temperature. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GSK3 beta (phospho Y216) + GSK3 alpha (phospho Y279) antibody [EPR933Y] (AB68476)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human glioma tissue sections labeling GSK3 (alpha + beta) with Purified ab68476 at 1 : 50 dilution (5.3 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1 : 0 dilution. PBS instead of the primary antibody was used as the negative control.

• IP

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Immunoprecipitation - Anti-GSK3 beta (phospho Y216) + GSK3 alpha (phospho Y279) antibody [EPR933Y] (AB68476)

GSK3 (alpha + beta) (phospho Y216 + Y279) was immunoprecipitated from 10μg HeLa (human cervix adenocarcinoma) whole cell lysate with ab68476 at 1/50 dilution (2μg in 0.35mg lysates). Western blot was performed from the immunoprecipitate using ab68476 at 1/200 dilution (9 μg/ml). VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Lane 1 (Input) : HeLa (human cervix adenocarcinoma) treated with 1uM staurosporine for 4h whole cell lysate 10μg
Lane 2 (+) : HeLa (human cervix adenocarcinoma) treated with 1uM staurosporine for 4h whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab68476 in HeLa (human cervix adenocarcinoma) treated with 1uM staurosporine for 4h whole cell lysate

Exposure Time : 30 seconds
Blocking and diluting buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-GSK3 beta (phospho Y216) + GSK3 alpha (phospho Y279) antibody [EPR933Y] (ab68476)

Observed band size: 47 kDa,51 kDa

false

Exposure time: 30s

• WB

Unknown

Western blot - Anti-GSK3 beta (phospho Y216) + GSK3 alpha (phospho Y279) antibody [EPR933Y] (AB68476)

All lanes:

Western blot - Anti-GSK3 beta (phospho Y216) + GSK3 alpha (phospho Y279) antibody [EPR933Y] (ab68476) at 1/2000 dilution

Lane 1:

HEK-293 (Human epithelial cell line from embryonic kidney) cell lysate, cells untreated at 10 µg

Lane 2:

HEK-293 cell lysate, cells treated with AP at 10 µg

Secondary

All lanes:

HRP conjugated Goat anti-rabbit at 1/2000 dilution

Observed band size: 47-52 kDa

false

• WB

Lab

Western blot - Anti-GSK3 beta (phospho Y216) + GSK3 alpha (phospho Y279) antibody [EPR933Y] (AB68476)

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

All lanes:

Western blot - Anti-GSK3 beta (phospho Y216) + GSK3 alpha (phospho Y279) antibody [EPR933Y] (ab68476) at 1/500 dilution

Lane 1:

SH-SY5Y (Human neuroblastoma epithelial cell) whole cell lysates at 15 µg

Lane 2:

SH-SY5Y (Human neuroblastoma epithelial cell) treated with nerve growth factor at 100ng/ml for 5 minutes. Whole cell lysates. at 15 µg

Lane 3:

SH-SY5Y (Human neuroblastoma epithelial cell) treated with nerve growth factor at 100ng/ml for 5 minutes. Whole cell lysates. Then the membrane was incubated with phosphatase. 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

Observed band size: 47-52 kDa

false

Exposure time: 10s

• Dot

Unknown

Dot Blot - Anti-GSK3 beta (phospho Y216) + GSK3 alpha (phospho Y279) antibody [EPR933Y] (AB68476)

Dot Blot analysis of Lane 1 : Human GSK3 (alpha + beta) (pY216 + pY279) phospho peptide and Lane 2 : Human GSK3 (alpha + beta) non-phospho peptide labeling GSK3 (alpha + beta) (phospho Y216 + Y279) with ab68476 at 1/1000 dilution. 5% NFDM/TBST was used as the diluting and blocking buffer. ab97051 Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated was used as the secondary antibody at 1/100000 dilution. Exposure time : 3 minutes.

• WB

Lab

Western blot - Anti-GSK3 beta (phospho Y216) + GSK3 alpha (phospho Y279) antibody [EPR933Y] (AB68476)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-GSK3 beta (phospho Y216) + GSK3 alpha (phospho Y279) antibody [EPR933Y] (ab68476) at 1/1000 dilution

Lane 1:

Mouse brain lysates. Then the membrane was incubated with phosphatase at 15 µg

Lane 2:

Untreated Mouse brain lysates 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

false

• WB

Supplier Data

Western blot - Anti-GSK3 beta (phospho Y216) + GSK3 alpha (phospho Y279) antibody [EPR933Y] (AB68476)

Blocking and dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-GSK3 beta (phospho Y216) + GSK3 alpha (phospho Y279) antibody [EPR933Y] (ab68476)

Lane 1:

PC12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate at 15 µg

Lane 2:

Whole cell lysate from PC12 (Rat adrenal gland pheochromocytoma cell line) cells treated with nerve growth factor-β at 100ng/ml for 5 minutes. at 15 µg

Lane 3:

Whole cell lysate from PC12 (Rat adrenal gland pheochromocytoma cell line) cells ) treated with nerve growth factor-β at 100ng/ml for 5 minutes. Membrane incubated with phosphatase 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

true

Exposure time: 3s

• WB

Lab

Western blot - Anti-GSK3 beta (phospho Y216) + GSK3 alpha (phospho Y279) antibody [EPR933Y] (AB68476)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-GSK3 beta (phospho Y216) + GSK3 alpha (phospho Y279) antibody [EPR933Y] (ab68476) at 1/1000 dilution

Lane 1:

Zebrafish lysates Then the membrane was incubated with phosphatase at 15 µg

Lane 2:

Untreated Zebrafish lysates 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

Observed band size: 47-52 kDa

false

• WB

CiteAb

Western blot - Anti-GSK3 beta (phospho Y216) + GSK3 alpha (phospho Y279) antibody [EPR933Y] (AB68476)

GSK3 beta (phospho Y216) + GSK3 alpha (phospho Y279) western blot using anti-GSK3 beta (phospho Y216) + GSK3 alpha (phospho Y279) antibody [EPR933Y] ab68476. Publication image and figure legend from Zhao, C., Yu, H., et al., 2022, Oncogenesis, PubMed 35606353.

ab68476 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 ab68476 please see the product overview.

GBM with ZDHHC4 knockdown in vivo was more sensitive to TMZ treatment.A U118R cells (shNC, shZDHHC4-1, and shZDHHC4-2, n = 5/group) (5 × 105 cells/mouse) were injected into nude mice. Mice were sacrificed 45 days later. H&E staining demonstrated typical tumor xenografts. B Intracranial tumor volumes in panel A were calculated (mean ± SD, n = 5 for each group, two-tailed Student’s t-test). C GSK3β palmitoylation and GSK3β-STAT3 pathway activity in tumors were detected by western blot. D The mRNA levels of GSC markers (OCT4, NANOG, CD133, and SOX2) in tumor tissues at the end of the experiment were analyzed by RT-PCR. The folding changes were normalized to shNC (mean ± SD, n = 5 for each group, two-tailed Student’s t-test). E U118R cells (shNC and shZDHHC4-2 each in two groups, n = 5/group) were injected into nude mice. Three days after cell injection, mice were intraperitoneally injected with TMZ (25 mg kg−1 d−1) every other day for 30 days. Mice were sacrificed humanely 45 days later. H&E staining demonstrated typical tumor xenografts. F Intracranial tumor volumes in (E) were calculated (mean ± SD, n = 5 for each group, two-tailed Student’s t-test). G The mice were weighed every 4 days (mean ± SD, n = 5 for each group, two-tailed Student’s t-test). H Kaplan–Meier survival curves were used to define the overall survival of intracranial tumor-bearing mice.

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