Bcl xl

Manufactured by Cell Signaling Technology

Sourced in United States, United Kingdom, Germany, China, Japan, Canada

Bcl-xL is a protein that plays a role in the regulation of apoptosis, or programmed cell death. It is a member of the Bcl-2 family of proteins and functions as an anti-apoptotic factor, helping to prevent cell death. Bcl-xL is commonly used in cell biology research to study the mechanisms of apoptosis and cell survival.

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Lab products found in correlation

752 protocols using bcl xl

Immunoprecipitation and Western Blot Analysis

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Total cell lysates were prepared in 1% CHAPS buffer [5mM MgCl2, 140 mMNaCl, 1mM EDTA, 1mM EGTA, 1% CHAPS, 20mM Tris-HCl (pH 7.5), and protease inhibitors (cOmplete ULTRA, Roche)]. AlgiMatrix dissolving buffer (ThermoFisher Scientific) was used to harvest spheroids before lysis in 1% CHAPS buffer. Proteins (600–1000 μg) were immunoprecipitated with BCL-2 (#4223, Cell Signaling), BCL-XL (#2762, Cell Signaling), MCL-1 (S-19, Santa Cruz) antibodies at 4°C for 16h and coimmunoprecipitates were captured by Dynabeads Protein G at 4°C for 2 h. Beads were recovered using DynaMag spin magnet and washed twice in 1% CHAPS buffer. Total cell lysates and immunoprecipitates were separated on NuPage 10% Bis-Tris gels. After SDS-PAGE, proteins were transferred onto PVDF membranes (Millipore) and then blocked with 5% dried milk in PBS-Tween20. Membranes were incubated with primary and secondary antibodies (GE Healthcare) in a buffer containing 10% milk diluent-blocking concentrate (KPL), detected with Luminata Crescendo Western HRP substrate (Millipore). Blots were imaged with LAS4000 image analyzer (Fujifilm) on chemiluminescence mode. The following antibodies were used for immunoblotting: BCL-2 (#2872, Cell Signaling), BCL-XL (#2762, Cell Signaling), Actin (#8457, Cell Signaling), BIK (#4592, Cell Signaling), MCL-1 (S-19, Santa Cruz), p53 (DO-1, Santa Cruz).

Kutuk O., Aytan N., Karakas B., Kurt A.G., Acikbas U., Temel S.G, & Basaga H. (2017). Biphasic ROS production, p53 and BIK dictate the mode of cell death in response to DNA damage in colon cancer cells. PLoS ONE, 12(8), e0182809.

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Western Blot Analysis of Splenocyte Proteins

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Whole cell lysates were made from splenocytes in a RIPA lysis buffer with PhosSTOP phosphatase inhibitor cocktail and complete Protease Inhibitor Cocktail (Roche Applied Science, Indianapolis, IN, USA). Proteins were separated on Novex^® NuPAGE^® sodium dodecyl sulfate-polyacrylamide gel (Invitrogen), transferred to polyvinylidene fluoride membranes and immunoblotted using the indicated antibodies and horseradish peroxidase–labeled secondary antibodies (GE Healthcare Biosciences, Pittsburgh, PA, USA). Blots were developed by chemiluminescence (Thermo Fisher Scientific), recorded on an LAS-4000 imaging system (Fujifilm Global, Valhalla, NY, USA), and quantified using Multi Gauge software (Version 3.1; Fujifilm). Antibodies to γ-Tubulin (Sigma), IκBα, Bcl-xL, Mcl-1 and GapdH (all from Cell Signaling Technology, Inc, Danvers, MA, USA), were used for immunoblotting.

Niemann C.U., Mora-Jensen H.I., Dadashian E.L., Krantz F., Covey T., Chen S.S., Chiorazzi N., Izumi R., Ulrich R., Lannutti B.J., Wiestner A, & Herman S.E. (2017). Combined BTK and PI3Kδ inhibition with acalabrutinib and ACP-319 improves survival and tumor control in CLL mouse model. Clinical cancer research : an official journal of the American Association for Cancer Research, 23(19), 5814-5823.

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Evaluating SNX-2112 Cytotoxicity and Signaling

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SNX-2112 was synthesized as previously described in our lab with >98.0% purity [42 (link)], dissolved in Dimethyl sulfoxide (DMSO) to obtain a 100 mM stock solution, and stored at −20°C. TRAIL was purchased from Merck Millipore (Waltham, MA, USA). N-Acetyl-cysteine (NAC) and 2′,7′-dichlorofluorescein diacetate (DCFH-DA) were purchased from the Beyotime Institute of Biotechnology (Shanghai, China). Bafilomycin A1 (BFA) was purchased from Selleck Chemicals (Shanghai, China). Dimethyl sulfoxide (DMSO) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma (St. Louis, MO, USA). Antibodies for GAPDH, Bcl-2, Bcl-xL, FLIP, pro-caspase 3, cleaved-caspase 3 (c-caspase 3), cleaved-caspase 8 (c-caspase 8), cleaved-PARP (c-PARP), Akt, p-Akt (Ser473), DR4, DR5, LC3, Beclin1, Atg7, p62, p-mTOR, p-S6, p-4EBP1, p53, p-ERK, ERK, p-p38, p38, p-JNK, and JNK were purchased from Cell Signaling Technology (CST; Beverly, MA, USA).

Hu L., Wang Y., Chen Z., Fu L., Wang S., Zhang X., Zhang P., Lu X., Jie H., Li M., Wang Y, & Liu Z. (2019). Hsp90 Inhibitor SNX-2112 Enhances TRAIL-Induced Apoptosis of Human Cervical Cancer Cells via the ROS-Mediated JNK-p53-Autophagy-DR5 Pathway. Oxidative Medicine and Cellular Longevity, 2019, 9675450.

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Bazedoxifene Modulates Colon Cancer Signaling

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Human colon cancer cell lines (DLD-1, HCT-15, and HCT-116) at 50–60% confluence were harvested after an overnight treatment with bazedoxifene or DMSO, and then lysed in cold RIPA lysis buffer containing protease inhibitor cocktail and phosphatase inhibitor cocktail. The lysates were subjected to 10% or 12% SDS-PAGE gel and transferred to a PVDF membrane. Membranes were probed with a 1:1000 dilution of specific primary antibody and 1:10,000 HRP-conjugated secondary antibody. Primary antibodies against phosphorylated STAT3 (Tyr705, p-STAT3^Y705), IL-6, BCL-XL, c-MYC, survivin, cyclin D1, STAT3, AKT, ERK, phospho-specific extracellular signal-regulated kinase (ERK) 1/2 (threonine 202/Tyrosine 204), phosphorylated-AKT (Ser473), GAPDH and secondary antibodies were all from Cell Signaling Technology (Beverly, MA, USA). Primary antibodies against IL-11, IL-11Rα and IL-6R were from Abcam (Cambridge, MA, USA). Membranes were analyzed using enhanced chemiluminescence plus reagents and scanned with the Storm Scanner (Amersham Pharmacia Biotech Inc., Piscataway, NJ).

Wei J., Ma L., Lai Y.H., Zhang R., Li H., Li C, & Lin J. (2019). Bazedoxifene as a novel GP130 inhibitor for Colon Cancer therapy. Journal of Experimental & Clinical Cancer Research : CR, 38, 63.

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Cell Proliferation and Apoptosis Assay

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A cell counting kit (CCK) for WST assay was purchased from Donginbiotech Co. (Seoul, Korea). Crystal violet solution was purchased from Sigma-Aldrich (St Louis, MO, USA). Anti-phospho-AMPK, phospho-p38, phospho-ERK, phospho-JNK, cyclin D1, CDK4, AMPK, PARP, caspase-3, Bcl-2, Bcl-xL, and Bax antibodies were purchased from Cell Signaling (Danvers, MA, USA). Anti-p38, ERK, JNK, GAPDH, and α-tubulin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Mun J.G., Jeon H.D., Yoon D.H., Lee Y.S., Park S.Y., Jin J.S., Park N.J, & Kee J.Y. (2022). Supercritical Extract of Cannabis sativa Inhibits Lung Metastasis in Colorectal Cancer Cells by Increasing AMPK and MAPKs-Mediated Apoptosis and Cell Cycle Arrest. Nutrients, 14(21), 4548.

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Western Blot Protein Analysis Protocol

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Proteins were extracted by boiling cell pellets in sample buffer (0.35 M Tris pH 6.8, 0.1 g ml⁻¹ sodium dodecyl sulphate, 93 mg ml⁻¹ dithiothreitol, 30% glycerol, 50 µg ml⁻¹ bromophenol blue), resolved by SDS-PAGE, then electroblotted onto Immobilon-P membranes (Merck Millipore). Following blocking in 5% dried skimmed milk (Marvel) dissolved in TBST (50 mM Tris pH 7.6, 150 mM NaCl, 0.1% Tween-20), membranes were incubated with primary antibodies (BCL-xL, Cell Signaling cat. no. 2762, RRID:AB_10694844; EGR1, Abcam cat. no. ab194357; GFP, Cell Signaling cat. no. 2956, RRID:AB_1196615; c-MYC, Abcam cat. no. ab32072, RRID: AB_731658; NOXA, CalbioChem cat. no. OP180, RRID:AB_2268468; PLK1, Cell Signaling cat. no. 4513, RRID:AB_2167409; SAE2, Abcam cat. no. ab185955; TAO1, in house) overnight at 4°C. Membranes were then washed three times in TBST and incubated for at least 1 h with appropriate horseradish-peroxidase-conjugated secondary antibodies (Invitrogen). After washing in TBST, bound secondary antibodies were detected using either EZ-Chemiluminescence Reagent (Geneflow Ltd) or Luminata™ Forte Western HRP Substrate (Merck Millipore) and a Biospectrum 500 imaging system (UVP) or ChemiDoc™ Touch Imaging System (BioRad).

Littler S., Sloss O., Geary B., Pierce A., Whetton A.D, & Taylor S.S. (2019). Oncogenic MYC amplifies mitotic perturbations. Open Biology, 9(8), 190136.

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Western Blot Analysis of EMT and Apoptosis Markers

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After the indicated treatment, cells were harvested and lysed in lysis buffer. Equal amount of proteins (10–30 μg) were separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Millipore). The membranes were then blocked with 5% non-fatty dry milk in TBST for 2 h and probed overnight at 4°C with primary antibodies against the following proteins: 14-3-3ζ (1/1000, Abcam), E-cadherin (1/1000, Cell Signaling Technology), vimentin (1/1000, Cell Signaling Technology), Snail (1/1000, Cell Signaling Technology), Slug (1/1000, Cell Signaling Technology), Bcl-2 (1/1000, Cell Signaling Technology), Bcl-xL (1/1000, Cell Signaling Technology), MCL-1 (1/1000, Cell Signaling Technology), Bax (1/1000, Cell Signaling Technology), PARP (1/1000, Cell Signaling Technology), cleaved caspase-3 (1/1000, Cell Signaling Technology) and β-actin (1/2000, Cell Signaling Technology). The membranes were washed in TBST and then incubated in HRP-conjugated secondary antibodies (1/5000, Cell Signaling Technology) for additional 1 h. The specific protein bands on the membranes were visualized by enhanced chemiluminescence (Millipore).

Wei L., Hu N., Ye M., Xi Z., Wang Z., Xiong L., Yang N, & Shen Y. (2022). Overexpression of 14-3-3ζ primes disease recurrence, metastasis and resistance to chemotherapy by inducing epithelial-mesenchymal transition in NSCLC. Aging (Albany NY), 14(14), 5838-5854.

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Immunoblotting of Cell Signaling Proteins

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Immunoblotting was performed with the following antibodies: Bax (D2E11, #5023), Bcl-xL (#2762), Caspase-3 (#9662), Caspase-9 (#9502), p-Cdc2 (Tyr15) (#9111), Cdc2 (POH1, #9116), p-CDK2 (Thr160) (#2561), p-Chk2 (Thr68) (C13C1, #2197), Chk2 (#2662), c-Raf (#9422), CDK2 (78B2, #2546), Cyclin D1 (#2922), Cyclin E1 (D7T3U, #20808), DUSP4 (D9A5, #5149), DUSP6 (#39441), p-Erk1/2 (Thr202/Tyr204) (D13.14.4E, #4370), Erk1/2 (137F5, #4695), Mcl-1 (D5V5L, #39224), MDM2 (D1V2Z, #86934), p-MEK1/2 (Ser217/221) (41G9, #9154), MEK1/2 (47E6, #9126), p21 (12D1, #2947), p-p53 (Ser15) (16G8, #9286), p53 (7F5, #2527), PARP (46D11, #9532), PUMA (D30C10, #12450), p-Rb (Ser789) (#9307), (all from Cell Signaling Technology (Boston, MA, USA)), Actin (C-2, #sc-8432 AF790, Santa Cruz Biotechnology), and β-actin antibody (AC-15, #A5441, Sigma-Aldrich). Immunoblotting signals were visualized using an Odyssey IR imaging system (Li-Cor Biosciences, Lincoln, NE, USA). Image Studio software v4.0 was used for analysis of the bands.

Pairawan S., Akcakanat A., Kopetz S., Tapia C., Zheng X., Chen H., Ha M.J., Rizvi Y., Holla V., Wang J., Evans K.W., Zhao M., Busaidy N., Fang B., Roth J.A., Dumbrava E.I, & Meric-Bernstam F. (2022). Combined MEK/MDM2 inhibition demonstrates antitumor efficacy in TP53 wild-type thyroid and colorectal cancers with MAPK alterations. Scientific Reports, 12, 1248.

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Cytotoxicity Evaluation of Compounds

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Dulbecco’s modified Eagle’s medium (DMEM), Hoechst 33,342, Fluoromount, 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA), and Rhodamine-123 and Fetal Bovine Serum (FBS) were purchased from Sigma (St. Louis, MO, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and trypsin were procured from Hi-media Pvt. Limited, Mumbai (India). Rabbit monoclonal Bcl-xl, p-53, p-NF-κB, Caspase3, and Caspase9 antibodies and anti-rabbit- HRP secondary antibody were obtained from Cell Signaling Technology, Danvers, MA, USA. PVDF membrane (MDI, Ambala). The RT-PCR chemical kit was purchased from Bio-Rad, CA, USA. The BD Cycletest plus DNA Kit was from BD Biosciences, San Jose, CA, USA. Chemicals and reagents of analytical (AR) grade were used to perform the experiments.

Kumar A., Kaur S., Dhiman S., Singh P.P., Bhatia G., Thakur S., Tuli H.S., Sharma U., Kumar S., Almutary A.G., Alnuqaydan A.M., Hussain A., Haque S., Dhama K, & Kaur S. (2022). Targeting Akt/NF-κB/p53 Pathway and Apoptosis Inducing Potential of 1,2-Benzenedicarboxylic Acid, Bis (2-Methyl Propyl) Ester Isolated from Onosma bracteata Wall. against Human Osteosarcoma (MG-63) Cells. Molecules, 27(11), 3478.

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Signaling Pathways in Cell Cycle Regulation

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The chemicals used in this study were crizotinib (Selleck Chemicals LLC, Houston, TX, USA), Cyclosporine A (CsA) (J&K chemical Ltd., Beijing, China), PD98059 (Selleck Chemicals LLC, Houston, TX, USA), MK-2206 (Selleck Chemicals LLC, Houston, TX, USA). The primary antibodies against phospho-Erk1/2, Erk1/2, phospho-AKT, AKT, phospho-STAT3, STAT3, phospho-Cdc25c, phospho-CDK1, CDK1, Cyclin B1, Bcl-XL, caspase-3 and PARP were purchased from Cell Signaling Technology (Boston, MA, USA). KSR2 and Cdc25c were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The secondary antibodies were horse radish peroxidase (HRP)-conjugated anti-rabbit IgG, anti-mouse IgG (Cell Signaling Technology).

Liu Z., Jiang L., Li Y., Xie B., Xie J., Wang Z., Zhou X., Jiang H., Fang Y., Pan H, & Han W. (2019). Cyclosporine A sensitizes lung cancer cells to crizotinib through inhibition of the Ca2+/calcineurin/Erk pathway. EBioMedicine, 42, 326-339.

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