Total protein lysates were obtained using RIPA buffer containing 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS), 1 mM EDTA, 50 mM Tris (pH 7.6), and 10 μL/mL protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO, USA). Equal amounts of protein from each sample were separated on SDS-PAGE gels and then transferred to nitrocellulose membranes. Membranes were blocked with 5% non-fat milk or 5% bovine serum albumin in TBS-T (150 mM NaCl, 10 mM Tris, pH 7.4, and 0.1% Tween-20), and incubated with the following primary antibodies at 4°C overnight: poly [ADP-ribose] polymerase (PARP), cleaved PARP, CHK1, phosphorylated CHK1 (Ser345), pH2A.X, and Rad51 (1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-CDK 4, anti-cyclin A, -B1, -D1, -E, p21CIP1, and cdc25A (1:250; Santa Cruz Biotechnology, Santa Cruz, CA, USA), active caspase 3 (1:500; Epitomics, Burlingame, CA), and anti-actin (1:3000; Millipore, Billerica, MA, USA) as a loading control. After several washes with TBS-T, the membranes were incubated with HRP-conjugated secondary antibody (1:5000; Bio-Rad, Hercules, CA, USA) for 1 h at room temperature. The bands were detected using an enhanced chemiluminescence detection system (GE Healthcare, Wauwatosa, WI, USA).
Active caspase 3
Manufactured by Abcam
Sourced in United States, United Kingdom
Active caspase 3 is a lab equipment product that functions as a key effector caspase involved in the execution phase of cell apoptosis. It plays a central role in the apoptotic process.
Automatically generated - may contain errors
Lab products found in correlation
Bcl 2, by Abcam (13 mentions)
Bcl2 associated x (bax), by Abcam (12 mentions)
P akt, by Abcam (7 mentions)
β actin, by Abcam (7 mentions)
Cyclin d1, by Abcam (4 mentions)
Pro caspase 3, by Abcam (4 mentions)
Bca protein assay kit, by Beyotime (4 mentions)
Gapdh, by Abcam (3 mentions)
α sma, by Abcam (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Active caspase 9, by Abcam (3 mentions)
Bcl 2, by Santa Cruz Biotechnology (3 mentions)
Gapdh, by Cell Signaling Technology (3 mentions)
P70s6k, by Abcam (3 mentions)
Histone h3, by Abcam (2 mentions)
Vimentin, by Abcam (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
Polyvinylidene fluoride (pvdf), by Thermo Fisher Scientific (2 mentions)
Muse cell analyzer, by Merck Group (2 mentions)
P cdk2, by Cell Signaling Technology (2 mentions)
50 protocols using active caspase 3
1
Western Blot Analysis of Cell Signaling
2
Western Blot Analysis of Apoptosis Markers
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Aliquots (30 μg of protein) of cell lysate were separated on a 12% SDS-PAGE gel, blotted into a nylon membrane, and probed with antibodies against caspase-3, active caspase-3, procaspase-8, caspase-9, and active caspase-9 (Epitomics, Inc.). Membranes were washed with 1% Tween 20 in tris buffered saline and incubated with a 1:3,000 dilution of HRP-conjugated secondary antibodies (Huaan, Hangzhou, People’s Republic of China) for 1 hour at room temperature. Protein bands were visualized by their enhanced chemiluminescence reaction.
3
Western Blot Analysis of Notch Signaling
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A cell lysis buffer system (Santa Cruz) was used to prepare the whole-cell extracts on dry ice. The Nuclear Extraction Reagents (Pierce) and Total Protein Extraction kit (Beyotime) were used according to the protocol provided by the manufacturers. The protein concentrations were detected with a BCA protein assay kit (Pierce). Vertical SDS-PAGE was used to separate the protein samples, which were then transferred to the PVDF membranes. After blocking with 10% defatted milk, primary antibodies against Delta-like 1 (DLL1, Sigma-Aldrich, 1: 2000), DLL3 (Sigma-Aldrich, 1: 2000), DLL4 (Sigma-Aldrich, 1: 2000), Jagged1 (Abcam, 1: 2500), Jagged2 (Abcam, 1: 2500), NICD (Cell Signaling Tech, 1: 4000), PI3K (Cell Signaling Tech, 1: 2000), Akt (Cell Signaling Tech, 1: 2000), phosphorylated Akt (p-Akt, Cell Signaling Tech, 1: 2000), Bcl2 (Abcam, 1: 2500), active caspase3 (Abcam, 1: 2000), GAPDH (Abcam, 1: 2000), and Histone H3 (Abcam, 1: 2000) were incubated at 4°C for 8 h. Then, the secondary antibodies were used to incubate the membranes at room temperature for 1 h. An ECL kit (Pierce) was used to develop the membranes, which were then exposed with Gene Genius (Syngene). Image J software was used to analyze the densities of the blots.
4
Quantifying Protein Expression in Cells
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Total proteins were quantified using BCA method (Beyotime Institute of Biotechnology, Shanghai, China). After that, proteins (30 μg proteins per lane) were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred onto polyvinylidene difluoride (PVDF) membrane (Thermo Fisher Scientific). After blocking with 5% skimmed milk in TBST for 1 h, the membrane was incubated with primary antibodies against α-SMA (1:1000, Abcam), Collagen III (1:1000, Abcam), Fibronectin (1:1000, Abcam), Vimentin (1:1000, Abcam), p-STAT1 (1:1000, Abcam), STAT1 (1:1000, Abcam), Fas (1:1000, Abcam), Active caspase 8 (1:1000, Abcam), Active caspase 3 (1:1000, Abcam), and GAPDH (1:1000, Abcam) at 4 °C overnight. Subsequently, the membrane was incubated with secondary antibodies (1:5000, Abcam) for 1 h at room temperature, and bands were detected with the ECL Chemiluminescent Substrate Reagent Kit (Thermo Fisher Scientific). GAPDH was acted as the internal control.
5
Hippocampal Protein Expression Analysis
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The ipsilateral hippocampi were isolated and homogenized in RIPA buffer containing a protease and phosphatase inhibitor cocktail (Roche, Indianapolis, IN, USA). The tissue homogenate was centrifuged at 12,000 rpm for 20 min at 4 °C and the supernatant was stored at −20 °C. Proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel and transferred to polyvinylidene difluoride membrane (Millipore). After blocking with 5% non-fat milk in phosphate buffer, membranes were incubated with primary antibodies overnight at 4 °C and subsequently incubated with secondary antibodies for 2 h at room temperature. Protein bands were visualized by enhanced chemiluminescence. β-Actin was used as a loading control. The following primary antibodies were used: active caspase-3 (abcam, 1:200), Arg1 (cell signaling, 1:1000), iNOS (Santa Cruz, 1:500), CD11b (Bioworld, 1:1000), β-Actin (cell signaling, 1:2000), p-Akt (cell signaling, 1:1000) and Akt (cell signaling, 1:1000). The data of western blot were quantified by Image J software.
6
Immunohistochemical Analysis of Kidney Tissue
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Paraffin-embedded kidney tissue sections (4 μm) were de-paraffinized, rehydrated with concentration gradient of ethanol. The sections were subjected to antigen retrieval step, by microwave-based method in 10 mM citrate buffer, or incubation with 20 ug/ml proteinase k (Qiagen, Valencia, CA, USA) for 10 min Then, the sections were quenched in 3% hydrogen peroxide for 10 min and incubated with 2% BSA to block nonspecific binding. Primary antibodies against β-catenin (BD Bioscience, San Jose, CA, USA), aquaporin-1 and active caspase-3 (Abcam) were added to the sections overnight and subsequently with secondary peroxidase conjugated antibodies (Dako) or alkaline phosphatase conjugated antibodies (Enzo Life Sciences, Farmingdale, NY, USA) for 1 h. The sections were developed by using DAB substrate from the Envision Plus system (Dako) or HIGHDEF red chromogen (Enzo Life Sciences), followed by counterstain with CAT hematoxylin (Biocare Medical, Walnut Creek, CA, USA). The slides were then dehydrated with ethanol and xylene before mounting.
7
Characterizing Calu-3 Spheroid Polarity and Apoptosis
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Briefly, cells were fixed and permeabilized using, respectively, 4% paraformaldehyde in PBS and 0.2 M NH4Cl/PBS containing 0.2% Triton X-100 for 10 min at room temperature. After blocking with 3% bovine serum albumin (BSA; Sigma-Aldrich) in PBS for 2 h, F-actin and the nuclei were stained with Phalloidin-TRITC (2 μg/mL; Sigma-Aldrich) and DAPI (1 μg/mL; Sigma-Aldrich) for 60 min at room temperature, respectively. To characterize Calu-3 spheroid polarity, cells were first incubated with rabbit polyclonal anti-ZO-1 (1:100 dilution; Zymed Laboratories Inc.) and mouse monoclonal anti-integrin β1 antibodies (1:500 dilution; Abcam) overnight at 4°C. To identify apoptosis within Calu-3 spheroids, cells were first incubated with rabbit polyclonal active Caspase-3 (1:200 dilution; Abcam) overnight at 4°C. After washing with PBS 3 times (5 min each), the cells were incubated with FITC-conjugated donkey anti-rabbit IgG and TRITC-conjugated donkey anti-mouse IgG (both 1:200 dilution; Jackson ImmunoResearch) for 1 h to detect ZO-1, active Caspase-3 and β1 integrin, respectively. Samples were lastly washed 3 times with PBS, followed by examination under a confocal microscope (LSM710, Zeiss, Germany).
8
Investigating Hepatoprotective Mechanisms
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LPS and D-GalN were purchased from Sigma. CYG is composed of Herba artemisiae, Salvia miltiorrhiza, and rhubarb and was purchased from Jiangyin Tianjiang Pharmaceutical Co., Ltd. OMT was purchased from Shaanxi Baoji Fangsheng Biological Development Co., Ltd., with a purity of >98%. Akt, p-Aktser473, FoxO3a, p-FoxO3a, Bim, Bax, Bcl-2, and active-caspase 3 antibodies were purchased from Abcam Corporation, USA. TRIzol was purchased from Shanghai Pufei Biotechnology Co., Ltd. PCR primers were designed and synthesized by Shanghai Ruiqiang Biotechnology Co., Ltd. The apoptosis detection kit was purchased from Biouniquer, USA.
9
Western Blot Analysis of Cell Signaling
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Primary antibodies, including anti-TGIF1 (Cat # ab52955), Bcl-2 (Cat # ab32124), Active-Caspase3 (Cat # ab32042), Bax (Cat # ab182733), AKT (Cat # ab8805), p-AKT (Cat # ab38449), Cyclin D1 (Cat # ab134175), and anti-tubulin (Cat # ab210797) were purchased from Abcam (Cambridge, UK). HRP sheep anti-rabbit/mouse secondary antibodies were obtained from PTG Company.
10
Immunohistochemical Analysis of Tumor Tissues
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Tumor tissues were fixed in 4% paraformaldehyde for 48 h and then embedded in paraffin. After that, tumor tissues were cut into 5 μm slices. Consecutive sections were analyzed by IHC using antibodies against BCL6 and active caspase 3 (Abcam). After incubating with the secondary antibody for 1 h at room temperature, the sections were observed with a microscope.
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