All lanes : 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
Lane 2 : HEK-293T transfected with DDDDK and mCherry-tagged mouse Bmi1 T275A mutant expression vector, whole cell lysate
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
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

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Developed using the ECL technique.

Predicted band size: 37 kDa
Observed band size: 43 kDa why is the actual band size different from the predicted?


Exposure time: 3 seconds

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.

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.

Flow cytometric analysis of 4% 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.

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.

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.

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.