Following treatment with various concentrations of caffeine, GC cells were lysed using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) on ice for 40 min to extract the proteins. The protein concentration of the supernatants was determined via Bradford assay. Lysates (20 µg/lane) were separated via 10% SDS-PAGE, transferred to 0.22 µm polyvinylidene fluoride membranes (Sigma-Aldrich; Merck KGaA), and incubated at 4°C overnight with the following primary antibodies at a working dilution of 1:1,000, all purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA): Anti-tubulin (cat no. 2148), anti-cyclin D1 (cat no. 2978), anti-cyclin dependent kinase (CDK)4 (cat no. 12790), anti-p21 (cat no. 2947), anti-Bcl-2 (cat no. 4223), anti-Bcl-2-associated death promoter (Bad; cat no. 9239), anti-Bcl-2-associated X protein (Bax; cat no. 5023), anti-Cyt-c (cat no. 11940), anti-caspase-9 (cat no. 9502), anti-cleaved caspase-9 (cat no. 20750), anti-caspase-3 (cat no. 9665), and anti-cleaved caspase-3 (cat no. 9664). Membranes were then incubated with mouse anti-rabbit secondary antibody (cat no. 3678; 1:1,000; Cell Signaling Technology, Inc.) at 4°C for 2.5 h. Autoradiograms were semi-quantified via densitometry using ImageJ software version 1.46r (National Institutes of Health, Bethesda, MD, USA).
Anti bcl 2 associated x protein bax
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-Bcl-2-associated X protein (Bax) is an antibody that recognizes the Bax protein, which is a member of the Bcl-2 protein family. The Bax protein plays a core role in the regulation of apoptosis, or programmed cell death.
Automatically generated - may contain errors
Lab products found in correlation
Anti bcl 2, by Cell Signaling Technology (2 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Anti caspase 3, by Cell Signaling Technology (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti cleaved caspase 9, by Cell Signaling Technology (1 mentions)
Anti tubulin, by Cell Signaling Technology (1 mentions)
Anti cyt c, by Cell Signaling Technology (1 mentions)
Anti cyclin d1, by Cell Signaling Technology (1 mentions)
Anti cyclin dependent kinase cdk 4, by Cell Signaling Technology (1 mentions)
Anti p21, by Cell Signaling Technology (1 mentions)
Anti caspase 9, by Cell Signaling Technology (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by Beyotime (1 mentions)
Membrane filter, by Merck Group (1 mentions)
Anti actin antibody, by Santa Cruz Biotechnology (1 mentions)
Anti poly adp ribose polymerase parp, by Cell Signaling Technology (1 mentions)
Anti noxa, by Cell Signaling Technology (1 mentions)
Anti hdac2, by Cell Signaling Technology (1 mentions)
Protease inhibitor mixture, by Merck Group (1 mentions)
Anti γh2ax, by BioLegend (1 mentions)
5 protocols using anti bcl 2 associated x protein bax
1
Caffeine Modulates Apoptosis Signaling in GC Cells
2
Western Blot Analysis of p53 Signaling Pathway
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Cells were lysed in 1× SDS sample buffer supplemented with the protease inhibitor mixture (Sigma‐Aldrich, St Louis, MO, USA). Equal amounts of protein (30 μg) were separated on SDS/polyacrylamide gels and then transferred onto membrane filters (Merck Millipore, Amsterdam, the Netherlands). After blocking with 5% non‐fat dry milk, the membranes were probed with anti‐p53 (Santa Cruz Biotechnology, Dallas, TX, USA), anti‐phospho‐p53 at Ser‐15 (Cell Signaling Technology, Danvers, CA, USA), anti‐acetyl‐p53 at Lys‐373/382 (Upstate, Lake Placid, NY, USA), anti‐p21WAF1 (Santa Cruz Biotechnology), anti‐Bcl‐2‐associated X protein (BAX; Cell Signaling Technology), anti‐NOXA (Cell Signaling Technology), anti‐HDAC2 (Cell Signaling Technology), anti‐poly (ADP‐ribose) polymerase (PARP; Cell Signaling Technologies), anti‐γH2AX (BioLegend, San Diego, CA, USA), anti‐ATM (Santa Cruz Biotechnology), anti‐phospho‐ATM at Ser‐1981 (Merck Millipore) or with anti‐actin antibody (Santa Cruz Biotechnology) followed by an incubation with horseradish peroxidase‐conjugated secondary antibodies (Invitrogen). Immunodetection was performed with enhanced chemiluminescence (ECL; GE Healthcare Life Science, Piscataway, NJ, USA).
3
Biochemical Assays for Cardiac Injury
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Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco-BRL (Grand Island, NY, USA). The kits used for the determination of serum lactate dehydrogenase (LDH) and creatine kinase isoenzyme-MB (CK-MB) content were obtained from Jiancheng Bioengineering Institute (Nanjing, China). MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], TTC (2,3,5-triphenyltetrazolium chloride), Evans blue, and compound C [AMP-activated protein kinase (AMPK) inhibitor] were purchased from Sigma-Aldrich (St. Louis, MO, USA). The primary antibodies were as follows: anti-AMPKα (Thr172; #2532), anti-phosphorylated AMPKα (p-AMPKα) (Thr172; #2531), anti-caspase-3 (#9662), anti-Bcl-2 associated X protein (Bax; #2772), anti-cytochrome c (#11940), anti-acetyl CoA carboxylase (ACC; #3676), anti-p-ACC (#11818), β-actin (#4970), and anti-Bcl-2 (#2870; all from Cell Signaling Technologies, Beverly, MA, USA). All these are rabbit monoclonal antibodies. The secondary antibody (anti-rabbit IgG-B; sc-53804) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The caspase-3 assay kit was purchased from Chemicon International, Inc. (Temecula, CA, USA). The fluorescent kit for Hoechst 33258 was obtained from Roche Diagnostics (Mannheim, Germany).
4
Western Blot Antibody Validation
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Anti-β-actin antibody (sc-47778, monoclonal, raised in mouse, 1:10,000) was from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), MTT, Hoechst 33258 and RNaseA were all purchased from Sigma-Aldrich. Anti-AKT (#9272, polyclonal, raised in rabbit, 1:1,000), anti-phospho (p)-AKT (ser473; #4058, monoclonal, raised in rabbit, 1:1,000), anti-human epidermal growth factor receptor 2 (Her2; #2165, monoclonal, raised in rabbit, 1:1,000), anti-epidermal growth factor receptor (EGFR; sc-03, polyclonal, raised in rabbit, 1:500), anti-c-Raf (#9422, polyclonal, raised in rabbit, 1:1,000), anti-B-cell lymphoma 2 (Bcl-2; #2876, polyclonal, raised in rabbit, 1:1,000) and anti-Bcl-2- associated X protein (Bax; #2772, polyclonal, raised in rabbit, 1:1,000) antibodies were from Cell Signaling Technology (Beverly, MA, USA). Anti-cyclin-dependent kinase 4 (Cdk4; sc-260, polyclonal, raised in rabbit, 1:500), and anti-prostate-specific antigen (PSA; sc-7316, monoclonal, raised in mouse, 1:500), anti-AR (sc-7305, monoclonal, raised in mouse, 1:500), anti-Hsp70 (sc-24; monoclonal, raised in mouse, 1:500), Hsp90 (sc-69703, monoclonal, raised in mouse, 1:500) and NKX-3.1 (sc-15022, polyclonal, raised in goat, 1:500) antibodies were purchased from Santa Cruz Biotechnology, Inc. The Annexin V fluorescein isothiocyanate (FITC) Apoptosis Detection kit was purchased from BD Pharmingen (San Diego, CA, USA).
5
Western Blot Analysis of Signaling Proteins
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The total protein was extracted from cell lysates and was separated by SDS‐PAGE. The protein was transferred to expanded polyvinylidene difluoride membranes (Pierce, Rockford, IL) with a Bio‐Rad Trans‐Blot system. After blocking, membranes were incubated with antibodies, including anti‐Ras (1:1000, Abcam, Cambridge, MA), anti–Janus kinase (JAK) (1:1000, Abcam), anti–phosphorylated JAK (1:1000, Abcam), anti‐PI3K (1:1000, Abcam), anti–phosphorylated PI3K (1:1000, Abcam), anti‐AKT (1:1000, Cell Signaling, Danvers, MA), anti–phosphorylated AKT (1:1000, Cell Signaling), anti‐Bcl‐2‐associated X protein (BAX) (1:1000, Cell Signaling), anti–cleaved caspase 3 (1:500, Cell Signaling), and anti–B‐cell lymphoma 2 (BCL‐2) (1:1000, Arigo Hsinchu, Taiwan, China), or anti‐GAPDH (1:5000, Sigma‐Aldrich Co, St. Louis, MO). Protein signals were obtained by ECL‐Western blotting system (AVEGENE CHEMX 400) and quantified by the ImageJ software (NIH, Bethesda, MD). GAPDH was used for the normalization of signal bands of other genes.
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