Phospho bcl 2

Manufactured by Cell Signaling Technology

Sourced in United States

Phospho-Bcl-2 is a product offered by Cell Signaling Technology. It detects phosphorylation of the Bcl-2 protein, which is involved in the regulation of apoptosis (programmed cell death).

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Lab products found in correlation

Close spelling variants of this product

Anti-phospho-Bcl-2-Thr56 Phospho-Bcl-2 (Ser70), Anti-phospho-Bcl-2 Bcl-2

8 protocols using phospho bcl 2

Western Blot Analysis of Apoptosis Regulators

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Cell pellets were lysed in RIPA buffer (Macgene Technology Ltd.) with 10 μl/ml protease inhibitor cocktail (P8340, Sigma) and 10 μl/ml phosphatase inhibitor cocktail (p0044, Sigma). The proteins in cell lysates were separated by electrophoresis on a 15% SDS–polyacrylamide gel, transferred to nitrocellulose membranes, immunostained, and visualized by enhanced chemiluminescence detection reagents (Applygen Technologies Inc., Beijing, China). Antibodies against Survivin, Bcl-2, Bcl-xL, P-Histone 3, phospho-Survivin, and phospho-Bcl-2 were purchased from Cell Signaling (Danvers, MA), and the antibody specific for phospho-Ser-62-Bcl-xL was purchased from Abcam (Cambridge, UK). The β-Actin antibody was purchased from Zsbio (Beijing, China).

Han T.L., Sha H., Ji J., Li Y.T., Wu D.S., Lin H., Hu B, & Jiang Z.X. (2021). Depletion of Survivin suppresses docetaxel-induced apoptosis in HeLa cells by facilitating mitotic slippage. Scientific Reports, 11, 2283.

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Ginsenoside-Mediated Cytoprotective Mechanisms

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Ginsenosides (Rb1, Rc, Rg2, and Rg3), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide (MTT; cat. M5655), z-VAD-FMK (cat. V116), bafilomycin A1 (cat. B1793), N-acetyl-l-cysteine (NAC; cat. A9165), and propidium iodide (PI; cat. P4170) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies against Atg5 (cat. sc-515347), Bcl-2 (cat. sc-7382), caspase-9 (cat. sc-133109), caspase-3 (cat. sc-7148), poly (ADP-ribose) polymerase 1 (PARP1; cat. sc-8007), and actin (cat. sc-376421) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The LC3 (cat. 2775), Beclin-1 (cat. 3738), and phospho-Bcl-2 (cat. 2827) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Dihydrorhodamine (DHR) 123 (cat. D23806) and acridine orange (cat. A3568) were obtained from Molecular Probes (Eugene, OR, USA) and Invitrogen (Carlsbad, CA, USA), respectively.

Kang K.A., Yao C.W., Piao M.J., Zhen A.X., Fernando P.D., Herath H.M., Song S.E., Cho S.J, & Hyun J.W. (2022). Anticolon Cancer Effect of Korean Red Ginseng via Autophagy- and Apoptosis-Mediated Cell Death. Nutrients, 14(17), 3558.

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Compound Treatment Induces Apoptosis

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Cells were seeded in T-25 flasks at ∼2.5 × 10⁵ cells per flasks in 4 ml of cell-specific media and exposed to compounds at 0.5 μM for 24 h, 48 h or 72 h. Protein was prepared from treated cells using RIPA lysis buffer (Sigma) and quantified using the Bradford protein assay with Bradford reagent (Sigma). Equal amounts of protein were loaded in all wells (15 μg). Proteins were separated by electrophoresis through a 4–12% NuPAGE Bis-Tris gel (Invitrogen) and subsequently transferred to Hybond-P membrane (GE Healthcare, Buckinghamshire, UK).
Detection was carried out using anti-human cyclin B1 (ref. H433; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), phospho-Bcl2 (ref. 2827S; Cell Signaling Technology, Boston, MA, USA), caspase-3 (ref. 9665; Cell Signaling), GAPDH (ref. 2118; Cell Signaling) and p53 (ref. ab28-100; Abcam, Cambridge, UK) primary antibodies. Secondary alkaline phosphatase-conjugated anti-rabbit IgG (ref. 7054; Cell Signaling) or anti-mouse IgG (ref 7056; Cell Signaling) antibodies were used for detection in conjunction with a chemifluorescent substrate (ECF substrate, ref. RPN5785; GE Healthcare). Bands were visualised and quantified using a Storm imaging system (GE Healthcare).

Stengel C., Newman S.P., Day J.M., Chander S.K., Jourdan F.L., Leese M.P., Ferrandis E., Regis-Lydi S., Potter B.V., Reed M.J., Purohit A, & Foster P.A. (2014). In vivo and in vitro properties of STX2484: a novel non-steroidal anti-cancer compound active in taxane-resistant breast cancer. British Journal of Cancer, 111(2), 300-308.

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B-Cell Lymphoma Protocol: Cellular Characterization

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Pfeiffer cell line derived from human diffuse large B cell lymphoma cells were purchased from ATCC, USA. B95–8 lymphoma cells and EBV-infected marmoset white cells are a gift from Prof. Yao (The First Affiliated Hospital of Medical School of Zhejiang University). Mouse monoclonal antibody (3E8) against human B7-H4 is a gift from Institute of Infectious Diseases affiliated Zhejiang University. Goat anti-rabbit IgG antibodies and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgM were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif., USA). Rabbit antibodies against Bcl-2, phospho-Bcl-2, Bax, caspase 3, cleaved caspase 3, caspase 9, cleaved caspase 9, NF-κB p65, phosphor-NF-κB p65, extracellular-regulated kinase 1/2(Erk1/2), phospho-Erk1/2, Akt, phosphor-Akt and HRP-conjugated rabbit anti-D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody were purchased from Cell Signaling Technology (Danvers, Mass., USA).

Jiang Y., Cai G., Lin J., Zhang J., Bo Z., Li Y., Wang C, & Tong Y. (2019). B7-H4 is highly expressed in aggressive Epstein-Barr virus positive diffuse large B-cell lymphoma and inhibits apoptosis through upregulating Erk1/2 and Akt signalling pathways. Infectious Agents and Cancer, 14, 20.

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Delineating Apoptotic Signaling Pathways

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PR-619 was obtained from MedChemExpress (Junction, NJ, USA) and cisplatin from Merck Millipore (Billerica, MA, USA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Merck Millipore. For Western blot analysis, antibodies against cleaved caspase-3 (#9661), cleaved caspase-8 (#9496), B cell lymphoma (Bcl)-2 (#15071), phospho-Bcl-2 (#2824), phospho-p53 (#9284), caspase-4 (#4450), phospho-Janus kinase (JNK) (#9255), and c-Myc (#18683) were procured from Cell Signaling Technology (Danvers, MA, USA). The antibodies against β-actin (#109) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, #100118) were supplied by GeneTex (Irvine, CA, USA); those against α-tubulin (sc-5286), P21 (sc-6264), P27 (sc-1641), and JNK (sc-571) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). For IHC, the USP14 antibody (#MA5-32821) was purchased from Invitrogen, the USP21 antibody (#17856-1-AP) was purchased from Proteintech (Chicago, IL, USA), and the c-Myc (#ab32072) antibody was purchased from Abcam (Cambridge, UK).

Hsu F.S., Lin W.C., Kuo K.L., Chiu Y.L., Hsu C.H., Liao S.M., Dong J.R., Liu S.H., Chang S.C., Yang S.P., Chen Y.T., Chang R.J, & Huang K.H. (2021). PR-619, a General Inhibitor of Deubiquitylating Enzymes, Diminishes Cisplatin Resistance in Urothelial Carcinoma Cells through the Suppression of c-Myc: An In Vitro and In Vivo Study. International Journal of Molecular Sciences, 22(21), 11706.

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Western Blot Analysis of Cell Signaling

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Cell lysates were separated with SDS-PAGE as described previously.²⁸ Proteins were detected using specific primary antibodies against p38 MAPK (#9212), phospho-p38 MAPK (Thr180/Tyr182; #9215), MK2 (#3042), Hsp27 (#2402), phospho-Hsp27 (Ser82; #2401), Cdk1 (#9116), phospho-Cdk1 (Tyr15; #9111), phospho-Histone H3 (Ser28; #9713), cyclin B1 (#4135), Cdc25c (#4688), phospho-Cdc25c (Ser216; #4901), PARP (#95425), Bcl-2 (#2870), phospho-Bcl-2 (Ser70; #2870), Bcl-X_L (#2764), and Mcl-1 (#5453, all from Cell Signaling Technology, Danvers, MA, USA). Antibodies against phospho-MK2 (Thr334; #ab51018) and β-tubulin (#ab11308) were from Abcam (Cambridge, UK). Antibodies against β-actin (#A5316) and GAPDH (#737179) were from Sigma-Aldrich and Santa Cruz Biotechnology, respectively. Secondary antibodies were from Cell Signaling Technology. Detection was performed using the Immobilion Western HRP Substrate Luminol-Peroxidase reagent (MerckMillipore, Billerica, MA, USA) and the ChemiDoc MP System (Bio-Rad, Hercules, CA, USA). Band intensities were quantified by Image-Lab (Bio-Rad).

Gurgis F., Åkerfeldt M., Heng B., Wong C., Adams S., Guillemin G., Johns T., Chircop M, & Munoz L. (2015). Cytotoxic activity of the MK2 inhibitor CMPD1 in glioblastoma cells is independent of MK2. Cell Death Discovery, 1, 15028-.

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Antibody Validation for Cell Signaling

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The antibodies against β-actin (#4967), FLAG-tag (#2044), PARP (#9532), total JNK (#9252), phospho-JNK (#9251), total c-Jun (#9165), phospho-c-Jun (#9261), Total MKK4 (#9152), phospho-MKK4 (#9151), Total MKK7 (#4172), phospho-MKK7 (#4171), total Akt (#4691), phospho-Akt (#4060), total Bcl2 (#2876), and phospho-Bcl2 (#2827), were purchased from Cell Signaling Technology. The anti-serum against IBV S protein and N protein were from rabbits immunized with bacterial expressed fusion proteins as previously described^{48 (link), 49 (link)}.

Fung T.S, & Liu D.X. (2017). Activation of the c-Jun NH2-terminal kinase pathway by coronavirus infectious bronchitis virus promotes apoptosis independently of c-Jun. Cell Death & Disease, 8(12), 3215.

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Immunoblotting and Ubiquitination Analysis

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As described previously, cells were lysed, sonicated and protein concentration was quantified by BCA analysis (Thermo Scientific, Rockford, IL). 50μg of protein lysate was resolved on SDS-PAGE, transferred to PVDF membrane, and blotted with specific primary and secondary antibodies (Sigma-Aldrich) accordingly. [15 (link)] For IP-Western, 500μg of protein lysate was incubated with Ubiquilin 1 tandem UBA (TUBE2) agarose (Boston Biochem, Cambridge, MA) in the IP buffer (25mM Tris•HCl, pH7.4, 150mM NaCl and 1% NP-40) on a rotation platform at 4ºC overnight. Ubiquitinated proteins were bound to the agarose, washed three times with IP buffer and subjected to immunoblotting. [55 (link), 56 (link)] Primary antibodies were used for Mcl-1, CDK9, actin (Santa Cruz Biotechnology, Dallas, TX), phospho-Pol II CTD at Ser2 and Ser5, Pol II CTD (Covance, Princeton, NJ), phospho-ERK1/2, ERK1/2, phospho-CDK9, phospho-Bcl-2, Bcl-xL (Cell Signaling Technology, Danvers, MA), Bcl-2 (Dako, Carpinteria, CA) and GAPDH (EMD Millipore, Billerica, MA). Densitometry of immunoreactive bands on immunoblots was measured with FluorChem E System (ProteinSimple, Santa Clara, CA), quantified and normalized with AlphaView (ProteinSimple).

Yeh Y.Y., Chen R., Hessler J., Mahoney E., Lehman A.M., Heerema N.A., Grever M.R., Plunkett W., Byrd J.C, & Johnson A.J. (2014). Up-regulation of CDK9 kinase activity and Mcl-1 stability contributes to the acquired resistance to cyclin-dependent kinase inhibitors in leukemia. Oncotarget, 6(5), 2667-2679.

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