Total protein was extracted and quanti ed. Twenty-ve micrograms of protein was separated by 10% g polyacrylamide gel electrophoresis (PAGE), and then transferred to a nitrocellulose membrane. Skim milk 5% was used to block non-speci c antigen binding, and then primary antibodies were added (RASSF1A, Abcam, ab23950; YAP, Cell Signaling Technology, #4912; pS127-YAP, Cell Signaling Technology, #13008; Cyclin A, Santa Cruz, sc-596; Cyclin B1, Proteintech, 55004-1-AP; Cyclin D1, Santa Cruz, sc-246; Cyclin E, Santa Cruz, sc-25303; CDK1, Wanleibio, WL02373; cleaved-caspase-3, Wanleibio, WL02348; Bcl-2, Wanleibio, WL01556; BAX, Proteintech, 50599-2-lg; p73, Wanleibio, WL01604; p53, Santa Cruz, sc-126; p21, Wanleibio, WL0362; AKT, Wanleibio, WL0003b; p-AKT, Wanleibio, WLP001a; ERK, Wanleibio, WL01864; p-ERK, Wanleibio, WLP1512; STAT3, Wanleibio, WL03207; p-STAT3, Wanleibio, WLP2412; NF-κB P65, Wanleibio, WL01980; β-actin, Santa Cruz, sc-47778; Histone H3, Wanleibio) and incubated at 4 °C overnight. Next, secondary antibodies were added and incubated at room temperature for 1 h, and nally exposed on X-ray lm.
Bcl 2
Manufactured by Wanlei
Sourced in China, United States
The Bcl-2 is a laboratory equipment product used for the detection and analysis of the Bcl-2 protein. Bcl-2 is a key regulator of apoptosis, or programmed cell death, and plays a crucial role in various biological processes. The Bcl-2 equipment allows for the investigation of Bcl-2 expression and its involvement in different cellular and physiological contexts.
Automatically generated - may contain errors
Lab products found in correlation
Bcl2 associated x (bax), by Wanlei (23 mentions)
Cleaved caspase 3, by Wanlei (12 mentions)
Caspase 3, by Wanlei (8 mentions)
P akt, by Wanlei (6 mentions)
Cyclind1, by Wanlei (6 mentions)
Bcl2 associated x (bax), by Proteintech (5 mentions)
β actin, by Wanlei (5 mentions)
E cadherin, by Wanlei (5 mentions)
P erk, by Wanlei (4 mentions)
Vimentin, by Wanlei (4 mentions)
N cadherin, by Wanlei (4 mentions)
Bca protein assay kit, by Beyotime (4 mentions)
Gapdh, by Proteintech (4 mentions)
Cyclin a, by Santa Cruz Biotechnology (3 mentions)
Nf κb p65, by Wanlei (3 mentions)
Rassf1a, by Abcam (3 mentions)
Histone h3, by Wanlei (3 mentions)
Stat3, by Wanlei (3 mentions)
P stat3, by Wanlei (3 mentions)
Cyclin e, by Santa Cruz Biotechnology (3 mentions)
34 protocols using bcl 2
1
Western Blot Analysis of Protein Expression
2
Protein Expression Analysis in ccRCC
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Proteins were extracted from ccRCC cell lines by using urea buffer (2 M Thiourea, 40% CHAPS, 40mM Tris-Base, 40mM DTT, 2% Pharmalyte). Equal amounts of proteins were electrophoresed at sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to PVDF membranes with cold transfer buffer. Membranes were blocked with 5% non-fat dry milk in TBS-T for 1 h at room temperature (RT), and incubated with corresponding primary antibodies at 4°C overnight. Antibodies used are listed as following: β-actin (1: 1000 dilution), Bcl-2 (1: 500 dilution), Bax (1: 500 dilution), E-cadherin (1: 500 dilution ), N-cadherin (1: 500 dilution), Vimentin (1: 500 dilution), cleaved-caspase3 (1: 500 dilution), cleaved-caspase 12 (1: 500 dilution), from Wanleibio, China and MAN1C1 (1:500 dilution; Abcam, USA). Secondary antibodies, goat anti-mouse-HRP and goat anti-rabbit-HRP, were diluted at 1:2000.
3
Apoptosis Markers in Renal Tissues
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Protein expression levels of Bax, Bcl-2, and cleaved caspase 3 (Wanlei Biotechnology, Shenyang, China) in renal tissues were determined by western blot analysis. Briefly, frozen kidney tissues were lysed with radioimmunoprecipitation assay lysis buffer mixed with phenylmethylsulfonyl fluoride (Beyotime Biotechnology, Shanghai, China). After detection of total protein concentrations with a bicinchoninic acid assay kit (Beyotime Biotechnology), samples with equal volumes of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes, which were incubated with primary antibodies of Bax (1 : 1000), Bcl-2 (1 : 500), and cleaved caspase 3 (1 : 1000) in Primary Antibody Dilution Buffer (Leagene Biotechnology, Beijing, China) overnight at 4°C. After washing, membranes were incubated with goat anti-rabbit secondary antibody (ZSGB-BIO, Beijing, China) at 37°C for 2 h. All protein bands were captured with Amersham Imager 600 software (GE, Boston, MA, USA) and quantified with ImageJ.
4
Immunofluorescence Labeling Protocol
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FITC/Cy3-labeled goat anti rabbit/mouse IgG antibody was purchased from the Beyotime Biotechnology (Beijing, China). Nrf2, Caspase3, and Bcl2 antibodies were purchased from Wanlei Biotechnology Co., Ltd. (Shenyang, China). Unless otherwise specified, all the reagents were purchased from Sigma-Aldrich (Shaanxi, China).
5
Marine-Derived Streptomyces Compounds Evaluated
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Mor B, Saq B1, Saq B, and Lan N (purity > 98%) were obtained from Dr. Xie (Shandong University, Weihai, China), who isolated them from marine-derived Streptomyces sp. OC1610.4. Doxorubicin was acquired from Sigma-Aldrich Crop. (St. Louis, MO, USA). The compounds were dissolved in dimethyl sulfoxide (DMSO). Antibodies against PI3K, AKT, phosphor-AKT (Ser473), NF-κB, Twist, and GAPDH were purchased from Cell Signaling Technology (CST, Inc., Beverly, MA, USA). Antibodies against Bax, Bcl-2, E-cadherin, N-cadherin, Vimentin, Slug, and Snail were purchased from WanLei Biotechnology (Shenyang, China). The Annexin V-FITC apoptotic detection kit, 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine (JC-1) staining kit, and 4′,6-diamidino-2-phenylindol (DAPI) staining solution were purchased from Beyotime Institute of Biotechnology (Shanghai, China).
6
Western Blot Analysis of Apoptosis Regulators
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HK-2 cells were lysed with RIPA lysis buffer on the ice box. Protein concentrations were detected with BCA protein assay kit (Beyotime Biotechnology, Shanghai, China). Proteins were separated by electrophoresis on 10% SDS-polyacrylamide gel, transferred on the polyvinylidene difluoride (PVDF) membrane (Millipore, Burlington, MA, USA), and blocked by 5% fat-free milk at room temperature for 2 hours. The membranes were covered with primary antibodies and incubated at 4°C overnight, Bcl-2, Bax, β-actin at 1:1000 (Wanleibio, Shenyang, China). Then, membranes were incubated with HRP-labeled secondary antibody (1:20,000) (Proteintech, Wuhan, China) at room temperature for 1h after being washed by TBST for three times. The interested proteins were detected by ECL chemiluminescence. The data of blot bands were analyzed with Image J software.
7
Protein Expression Analysis via Western Blot
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Western blot was used to detect the expression of protein. Membranes were blocked with 5% TBST diluted nonfat powdered milk at room temperature for 1 hour incubated with primary antibody (HMGB1, sc-56,698, santa cruz; TLR4S, sc-293,072 santa cruz; Phosphorylation of NF(P-NF)-κB p65, sc-166,748 santa cruz; Cleaved caspase3, #9961, Cell Signaling Technology; NF-κB p65 #8242, Cell Signaling Technology; bcl-2, WL01556, Wanlei; Bax WL01637 Wanlei; GAPDH WL01114; β-actin WL01372 Wanlei.) at 4 C for 14 h. β-actin and GAPDH were used as the standard proteins. The secondary antibody was diluted with blocking solution and incubated membranes for 90 mins at room temperature [29 (link)]. Image J was used to analyze the gray value of the protein band.
8
Immunoblotting Antibody Validation Protocol
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VP was purchased from Sigma (St Louis, MO). Antibody against PML/RARα was purchased from Abcam (China). Antibodies against MST1, LATS1, YAP, p-YAP (S127), p38 MAPK, ERK, p-ERK (Thr202/Tyr204), AKT, p-AKT (S473), p-S6 (S235/236), c-Myc and caspase3 were from Cell Signaling Technology (USA), Antibodies against PARP, Survivin, Bcl-2, Bax, connective tissue growth factor (CTGF) and cyclinD1 were from Wanleibio (China). The antibody against p-p38 MAPK (Thr180/Tyr182) was purchased from Millipore (USA). Goat anti-rabbit antibody, goat anti-mouse antibody and antibody against β-Actin were purchased from Zhongshan Goldenbrige Biotechnology Co., Ltd (China).
9
Peiminine Modulates Autophagy and Apoptosis
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Antibodies targeting the following proteins were used in this study: cyclin D1 (WL01435a), CDK2 (WL01543), p27 (WL04174), cleaved caspase-9 (WL01838), cleaved caspase-3 (WL01992), Bcl-2 (WL01556), Bax (WL01637), LC3BII/I (WL01506), beclin-1 (WL02508), p62 (WL02385), p-JNK (WL01813), JNK (WL01295), c-JUN (WL02863), E-cadherin (WL01482), vimentin (WL01960), MMP-2 (WL1579), MMP-9 (WL01580), β-actin (WL01372), goat anti-rabbit IgG-HRP (WLA023) (all from Wanleibio, China), and p-c-JUN (AF3095, Affinity, United States). Purified peiminine (98%) was obtained from Yuanye Bio Co., Ltd (Shanghai, China; B20081). Cell Counting Kit-8 (CCK-8; C0037), the live/dead assay kit, the cell cycle and apoptosis analysis kit, the ROS Kit, NAC, SP600125, and the terminal dUTP nick-end labeling (TUNEL) Kit (C1086) were acquired from Beyotime Institute of Biotechnology (Shanghai, China). Tetramethylrhodamine methyl ester (TMRM; I34361) and Hoechst 3342 (H3570) were sourced from Life Technology (OR, United States). The mRFP-GFP-LC3 adenovirus was acquired from HanBio Technology Co., Ltd (Shanghai, China). The rest of the reagents and experimental materials were purchased from common commercial sources.
10
Protein Expression Analysis by Western Blot
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After a 48-hour transfection period, the cells were collected, washed twice with PBS, added to radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology, Shanghai, China) for 30 min on ice and centrifuged at 12,000 rpm for 15 min at 4°C. The total protein extracted was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking in 5% skim milk for 2 h, the membrane was incubated with primary antibodies at 4°C overnight. Next, the membrane was incubated with horseradish peroxidase-conjugated goat anti-ribbit secondary antibodies at 1:2,000 for 2 h. The proteins were visualized by BeyoECL Star (Beyotime Biotechnology). The primary antibodies for ZEB1, E-cadherin and N-cadherin were purchased from Cell Signaling Technology (Massachusetts, MA, USA). The primary antibodies for Bcl-2, Bcl-xL, Bax, caspase-3, caspase-8 and caspase-9 were purchased from Wanleibio (Shenyang, China).
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