Primary antibody against B cell leukemia/lymphoma 2 (Bcl-2) was obtained from Boster (Wuhan, Hubei, China). Primary antibodies against polyclonal antibodies against proliferating cell nuclear antigen (PCNA), Bcl-2 associated X (Bax), survivin, matrix metallopeptidase 9 (MMP9), matrix metallopeptidase 2 (MMP2), β-Actin and normal rabbit or mouse immunoglobulin G (IgG) were obtained from Proteintech (Wuhan, Hubei, China). Primary antibody against Notch4 was obtained from Santa Cruz Biotechnology (Dallas, TX, United States). Primary antibodies against P38/p-P38 MAPK, JNK/p-JNK, ERK/p-ERK were obtained from Cell Signaling Technology (Danvers, MA, United States). HRP-conjugated anti-Rabbit IgG and HRP-conjugated anti-mouse IgG were obtained from Servicebio (Wuhan, Hubei, China). HRP-conjugated anti-Rabbit IgG light chain and HRP-conjugated anti-mouse IgG light chain were obtained from Abbkine Scientific (Redlands, CA, United States). Protein G magnetic beads were obtained from Cell Signaling Technology (Danvers, MA, United States). U0126, SP600125 and SB203580 were obtained from MedChemExpress (Monmouth Junction, NJ, United States).
Bcl 2
Manufactured by Boster Bio
Sourced in China, United States, United Kingdom
Bcl-2 is a protein that plays a role in regulating apoptosis, or programmed cell death. It is a member of the Bcl-2 family of proteins and can act to inhibit apoptosis, thereby promoting cell survival.
Automatically generated - may contain errors
Lab products found in correlation
Bcl2 associated x (bax), by Boster Bio (21 mentions)
Caspase 3, by Boster Bio (6 mentions)
β actin, by Boster Bio (6 mentions)
Cleaved caspase 3, by Cell Signaling Technology (5 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (4 mentions)
Bcl2 associated x (bax), by Proteintech (3 mentions)
Ethidium bromide, by Merck Group (3 mentions)
Lc3 antibody, by Proteintech (3 mentions)
Hrp conjugated secondary antibody, by Boster Bio (3 mentions)
Ripa buffer, by Beyotime (2 mentions)
Caspase 9, by Boster Bio (2 mentions)
E cadherin, by Cell Signaling Technology (2 mentions)
Caspase 3, by Proteintech (2 mentions)
Di 2 pyridylketone, by Merck Group (2 mentions)
Rpmi 1640, by Merck Group (2 mentions)
Nf κb p65, by Boster Bio (2 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Gapdh, by Boster Bio (2 mentions)
35 protocols using bcl 2
1
Investigating Apoptosis and Signaling Pathways
2
Western Blot Analysis of ROS1 and EMT Markers
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The cells were lysed in RIPA buffer (Beyotime, Haimen, China) containing 1% protease inhibitor PMSF (Beyotime) and centrifuged at 12,000 rpm for 10 min. The supernatant containing total proteins was aspirated and the protein concentration was determined. The total proteins were separated by SDS-PAGE (Beyotime) and then transferred onto PVDF membranes (EMD Millipore, Billerica, MA, USA). After blocking, the membranes were incubated with primary antibodies against ROS1 (1:500, Sangon Biotech, Shanghai, China), cleaved-caspase-3 (1:1000, Abcam, Cambridge Science Park, Cambridge, UK), Bcl-2 (1:400, BOSTER, Wuhan, China), Bax (1:400, BOSTER), cleaved-PARP (1:1000, Abcam), E-cadherin (1:400, BOSTER), Vimentin (1:500, BIOSS, Beijing, China), N-cadherin (1:400, BOSTER), p-PI3K (1:500, BIOSS), PI3K (1:400, BOSTER), p-Akt (1:200, Santa Cruz Biotechnology, Dallas, Texas, USA) and Akt (1:200, Santa Cruz Biotechnology) at 4°C overnight. After washing with TBS-Tween 20 buffer, the membranes were incubated with goat anti-rabbit IgG-HRP (Beyotime) at 37°C for 45 mins. The bands were developed using ECL solution (Beyotime).
3
Oxidative Stress-Induced Apoptosis Signaling
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β-HB was obtained from Sigma Aldrich (St. Louis, MO, USA). Antibodies directed against caspase-12, phospho-p38 MAP kinase, phospho-SAPK/JNK, p38 MAP kinase, SAPK/JNK, Phospho-ERK1/2, ERK1/2 were obtained from Cell Signaling Technology (Danvers, MA, USA). AIF and ENDOG antibodies were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Antibodies directed against β-actin were obtained from Sungene Biotech (Nanjing, Jiangsu, China). Antibodies directed against PCNA, caspase-9, caspase-3, bax and bcl-2 were obtained from Boster Biotechnology (Wuhan, Hubei, China). Antibody directed against p53 was obtained from Keygen Biotech (Nanjing, Jiangsu, China). Fura-3/AM and BAPTA/AM were obtained from Dojindo Laboratories (Mashikimachi, Japan). NAC was obtained from beyotime institute of biotechnology (Haimen, Jiangsu, China). All the other reagents, unless otherwise stated, were purchased from Sigma Aldrich (St. Louis, MO, USA).
4
Protein Quantification and Western Blot Analysis
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The cells or tissues from in vitro or in vivo experiments were lysed in RIPA Buffer (1 mM EDTA pH 8.0, 50 mM Tris-HCl pH 8.0, 2% SDS, 5 mM DTT), and their protein concentration was determined by the BCA assay (Beyotime, Shanghai, China). The total protein (100 μg) was separated by 10%-15% SDS-PAGE gel and transferred to PVDF (polyvinylidene fluoride) membranes (Invitrogen, California, USA), and blocked with 5% skimmed milk powder in PBST (Phosphate Buffered Saline with Tween) or 5% w/v BSA, 1×TBS, 0.1% Tween at 4°C with gentle shaking about 12 hours. The membranes were immunoblotted with the primary antibodies for 4 hours at room temperature or overnight at 4°C and then incubated with the secondary antibodies for 2 hours. The corresponding semi-quantitative analysis was based on optical density with Image J software. In western blot analysis, cyclin B1, cyclin D1, BCL-2 (Boster Biological Technology), cyclin A, BAX, PARP, caspase-3, Vimentin (Proteintech), GAPDH (Bioworld), RRM2, E-cadherin, N-cadherin, phospho-AKT (T308), and phospho-ERK1/2 (T202/Y204) (Cell Signaling) were used for immunoblotting with secondary antibodies conjugated with horseradish peroxidase (Bioworld) and visualized with Bio-Rad ChemiDoc™ Imagers. All other chemical reagents were bought from Sigma (Shanghai, China).
5
Immunohistochemical Evaluation of Apoptosis Markers
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Tissue sections were dewaxed and rehydrated in xylene and ethanol solutions. Endogenous peroxidase was blocked by hydrogen peroxide. Antigen retrieval of these sections was performed in a 0.4 mol/L sodium citrate buffer through microwave after incubation with cleaved‐caspase3 (1:100; Cell Signaling Technology, Danvers, MA), Bax, Bcl‐2 (1:50, Boster, Wuhan, China), LC3 (1:50, Millipore, Billerica, MA), P62 (1:100, Sigma, St. Louis, MO), RIP, and smooth muscle α‐actin (1ː100, Abcam, Cambridge, UK) at 4°C overnight. All sections were labeled by goat antirabbit antibody (ZSGB‐BIO, Beijing, China) and visualized using a DAB Kit (ZSGB‐BIO, Beijing, China). The immunoreactivity in the pulmonary arterial medial layer was analyzed in a blinded method using an image‐processing program (Image‐Pro Plus, Version 6.0, Media Cybernetics, Rockville, MD).
6
Cell Viability and Apoptosis Assay
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MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), ethidium bromide (EB), di-2-pyridylketone, RPMI-1640, and other chemicals were purchased from Sigma-Aldrich (Shanghai, China). LC3 antibody was obtained from Proteintech Group (Wuhan, China). Antibodies against cyclin D1, caspase 3, β-actin, Bax, and Bcl-2 were purchased from Boster (Wuhan, China).
7
Western Blot Analysis of Apoptosis Markers
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Total cellular or tissue protein was extracted using RIPA lysis buffer. Proteins were separated using electrophoresis, transferred onto a polyvinylidene fluoride membrane, and separately incubated with the following primary antibodies: GAPDH (AC002, 1:5000, Abclonal, Wuhan, China), Caspase-3 (9662s, 1:1000, Cell Signaling Technology), Cleaved Caspase-3 (9664T, 1:1000, Cell Signaling Technology), Bcl-2 (BA0412, 1:200, Boster, Wuhan, China), Bax (5023T, 1:1000, Cell Signaling Technology). The membranes were washed and incubated with the corresponding secondary antibody (HRP-goat anti-rabbit IgG or goat anti-mouse antibody, 1:10000) for 1 h at room temperature. Proteins were visualized using enhanced chemiluminescence and the G: BOX Chemi X system (Syngene) per the manufacturer’s guidelines.
8
Western Blot Analysis of Cellular Proteins
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The lung tissues or treated A549 cells were harvested, and the whole proteins were extracted using a mixture of 100 µl RIPA lysate, 2 µl protease inhibitor (50×), and 1 µl 100 mM phenylmethanesulfonyl fluoride (PMSF, 100 mM). The protein concentration was determined using the bicinchoninic acid (BCA) method. Proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA, USA) and transferred to PVDF membranes. Membranes were blocked in 5% non-fat milk in TBST for 1 h at 37°C and probed with primary antibodies overnight at 4°C. After three washes, the membranes were incubated with the corresponding secondary antibodies for 1 h at 37°C. Signals were detected with ECL western blot detection reagents (Millipore, Billerica, MA, USA) using a direct chemiluminescent detection system (Fusion 182Fx5, Vilber, France). Protein expression was quantitated using ImageJ software (NIH, Bethesda, MD, USA). The primary antibodies used were: mTOR (Cell Signaling Technology, USA), STAT3, Bcl-2 (BOSTER Biological Technology, China), cyclin D1 (Beyotime, Shanghai, China), β-actin (ZSGB-BIO, China), and GRP78 (a kind gift from Prof. Deqiang Wang, Chongqing Medical University, Chongqing, China).
9
HEV RNA Induced Protein Expression
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The protein concentration from HEV RNA inoculated cells and controls were determined with the BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) and 20 μg of protein was separated by SDS-PAGE. The blots were incubated with anti-NOX4 (1:600), ATP5A1 (1:500), Bax (1:800), Bcl-2 (1:800), Caspase-9 (1:500), Caspase-3 (1:500) were purchased from Boster Biological Technology Co., Ltd. (China), Beijing Bioss Biological Technology Co., Ltd. (China) or ABclonal Biotechnology Co., Ltd (US), and β-actin (1:2000) purchased from Cell Signaling Technology (CST) (US), served as internal control, for 16 h at 4°C. The membranes were then incubated with the secondary antibodies conjugated with horseradish peroxidase (Santa Cruz Biotechnology) and developed by enhanced chemiluminescence (Thermo Scientific). The images were analyzed with ImageJ (NIH, Bethesda, MD, USA) Gray value was analyzed with ImageJ (NIH, Bethesda, MD, USA) to quantitatively analyze the expression levels of targeted proteins according to the level of exposure gray (resolution). Data were finally normalized to the expression of anti-β-actin.
10
Immunohistochemical and Western Blotting Assays
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Immunohistochemical staining and western blotting assays were performed using previously described methods [29 (link)]. The relative expression of protein was analyzed using Image J software with FracLac plugin java 1.6. Anti-YAP1 rabbit monoclonal antibody and anti-LATS1 rabbit monoclonal antibody were purchased from Proteintech. Anti-phospho-YAP1(Ser127) and anti-phospho-YAP1(Ser397) and anti-phospho-LATS1(S909) and HRP-linked secondary antibodies were purchased from Cell Signaling Technology, China. Anti-β-actin was purchased from TransGen Biotech (Beijing, China). Bcl-2 and Bax rabbit monoclonal antibody were purchased from Boster, China. Smad2/3 and p-Smad2/3 rabbit monclonal antibody were purchased from Proteintech (Wuhan, China).
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