Anti-Bmi1 (phospho T275) 抗体 [EPR19848]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Bmi1 (phospho T275) antibody [EPR19848] (AB213723)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized U-2 OS (human bone osteosarcoma epithelial cell line) cells labeling Bmi1 (phospho T275) with ab213723 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing increased nuclear foci staining observed in UV-treated U-2 OS cells. The cells were treated with 50 J/m² UV, then cultured in McCoy's 5a media supplemented with 10% FBS for 2 hours.

The nuclear counterstain is DAPI (blue). Tubulin is detected with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1/200 dilution (red).

The negative controls are as follows :
-ve control 1 : PBS instead of primary antibody (non-treated cells), followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.
-ve control 2 : PBS instead of primary antibody (UV treated cells), followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Bmi1 (phospho T275) antibody [EPR19848] (AB213723)

Intracellular flow cytometric analysis of4% paraformaldehyde-fixed, 90% methanol-permeabilized HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cell line transfected with DDDDK and mCherry-tagged mouse Bmi1 T275A mutant expression vector (left), or DDDDK and mCherry-tagged mouse Bmi1 WT expression vector (right) labeling Bmi1 (phospho T275) with ab213723 at 1/500 (red) compared with a Rabbit monoclonal IgG (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (black).

Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077),at 1/2000 dilution was used as the secondary antibody.

The cells were gated on the mCherry positive population.

The plasmids were kindly provided by Dr. Wei Guo from Tsinghua University.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Bmi1 (phospho T275) antibody [EPR19848] (AB213723)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK-293T cells (human epithelial cell line from embryonic kidney transformed with large T antigen) transfected with DDDDK and mCherry-tagged mouse Bmi1 (WT) expression vector labeling Bmi1 (phospho T275) with ab213723 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining in HEK-293T cells transfected with DDDDK and mCherry-tagged mouse Bmi1 (WT) expression vector. No staining was observed in HEK-293T cells transfected with DDDDK and mCherry-tagged mouse Bmi1 T275A mutant expression vector.

The nuclear counterstain is DAPI (blue). Tubulin is detected with Anti-Tubulin antibody [YOL1/34] - Microtubule Marker (Alexa Fluor^® 647) (ab195884) at 1/200 dilution (white).

The negative controls are as follows :
-ve control 1 : PBS instead of primary antibody (HEK-293T transfected with wild-type BMi1), followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.
-ve control 2 : PBS instead of primary antibody (HEK-293T transfected with Bmi1 T275A construct), followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Plasmids were kindly provided by Dr. Wei Guo from Tsinghua University.

• IP

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Immunoprecipitation - Anti-Bmi1 (phospho T275) antibody [EPR19848] (AB213723)

Bmi1 (phospho T275) was immunoprecipitated from 0.35 mg of HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) (transfected with DDDDK and mCherry-tagged mouse Bmi1 (WT) expression vector, followed by treatment with 800 J/m² UV, then cultured in DMEM containing 10% FBS for 30 minutes) whole cell lysate with ab213723 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab213723 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.

Lane 1 : HEK-293T (transfected / UV treated) whole cell lysate 10 μg (Input).

Lane 2 : ab213723 IP in HEK-293T (transfected / UV treated) lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab213723 in HEK-293T (transfected / UV treated) whole cell lysate.

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

Exposure time : 10 seconds.

All lanes:

Immunoprecipitation - Anti-Bmi1 (phospho T275) antibody [EPR19848] (ab213723)

Predicted band size: 36 kDa

Observed band size: 43 kDa

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• WB

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Western blot - Anti-Bmi1 (phospho T275) antibody [EPR19848] (AB213723)

Blocking and dilution buffer : 2% BSA/TBST

The phosphorylation of Bmi1 at T275 is increased by UV treatment.

The expression plasmids were kindly provided by Dr. Wei Guo from Tsinghua University.

All lanes:

Western blot - Anti-Bmi1 (phospho T275) antibody [EPR19848] (ab213723) at 1/2000 dilution

Lane 1:

HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) transfected with DDDDK and mCherry-tagged mouse Bmi1 (WT) expression vector, whole cell lysate at 20 µg

Lane 2:

HEK-293T transfected with DDDDK and mCherry-tagged mouse Bmi1 T275A mutant expression vector, whole cell lysate at 20 µg

Lane 3:

HEK-293T transfected with DDDDK and mCherry-tagged mouse Bmi1 (WT) expression vector, followed by treatment with 800 J/m² UV, then cultured in DMEM containing 10% FBS for 30 minutes, whole cell lysate at 20 µg

Lane 4:

HEK-293T transfected with DDDDK and mCherry-tagged mouse Bmi1 T275A mutant expression vector, followed by treatment with 800 J/m² UV, then cultured in DMEM containing 10% FBS for 30 minutes, whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 36 kDa

Observed band size: 43 kDa

true

Exposure time: 3s

• Dot

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Dot Blot - Anti-Bmi1 (phospho T275) antibody [EPR19848] (AB213723)

Dot blot analysis of Bmi1 (phospho T275) labeled with ab213723 at 1/1,000 dilution.

Lane 1 : Bmi1 (phospho T275) peptide.

Lane 2 : Bmi1 non-phospho peptide.

Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100,000 dilution was used as secondary antibody.

Blocking and dilution buffer : 5% NFDM/TBST.

Exposure time : 3 minutes.