All lanes : Anti-Bcl-2 (phospho S70) antibody [EPR21162] (ab218123) at 1/5000 dilution

Lane 1 : Untreated Jurkat (human T cell leukemia cell line from peripheral blood) whole cell lysate
Lane 2 : Jurkat treated with 1 µM paclitaxel for 24 hours 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

Predicted band size: 26 kDa


Exposure time: 32 seconds

Blocking/Dilution buffer and concentration: 2% BSA/TBST

The expression profile observed is consistent with what has been described in the literature (PMID: 10097113).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling Bcl-2 (phospho S70) with ab218123 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/100 dilution (green). Confocal image showing positive staining in HeLa cells in M phase. The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab195889) at 1/200 dilution (red). Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized Jurkat (human T cell leukemia T lymphocyte) treated with 1 μM paclitaxel for 24h (Right) / Untreated control (Left) cell line labeling Bcl-2 (phospho S70) with ab218123 at 1/500 dilution. Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

Square gate shows Bcl-2 (phospho S70) positive signal. The expression profile is consistent with what has been described in the literature (PMID: 10097113)

Flow cytometric analysis of 80% methanol-fixed, 0.1% Tween-20 permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cell line labeling Bcl-2 (phospho S70) with ab218123 at 1/500 dilution (right panel) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (left panel). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

Cells were pretreated with 20 μg/ml RNase A for 30 minutes to eliminate the non-specific binding between RNA and propidium iodide (PI).

Bcl-2 (phospho S70) is highly expressed in mitotic cells (PMID: 10567572).

Bcl-2 (phospho S70) was immunoprecipitated from 0.35 mg of Jurkat (human T cell leukemia cells from peripheral blood) treated with 1 μM paclitaxel for 24 hours whole cell lysate with ab218123 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab218123 at 0.5 μg/ml. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

Lane 1: Jurkat treated with 1 μM paclitaxel for 24 hours whole cell lysate 10 µg (Input). 

Lane 2: ab218123 IP in Jurkat treated with 1 μM paclitaxel for 24 hours whole cell lysate. 

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab218123 in Jurkat treated with 1 μM paclitaxel for 24 hours whole cell lysate.

Blocking/ Dilution buffer and concentration: 5% NFDM/TBST. Exposure time: 15 seconds.

Dot blot analysis of Bcl-2 (phospho S70) labeled with ab218123 at 1/1000 dilution.

Lane 1: Bcl-2 (phospho S70) peptide.
Lane 2: Bcl-2 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: 2% BSA/TBST.

Exposure time: 58 seconds.