Western blot was conducted as previously described [23 (link)]. Primary antibodies against Cyclin-D, p-Rb, Rb, Capase 3, p-STAT3, STAT3, LMNB1, p-JAK, JAK, p-Raf, Raf, p-MEK, MEK, p-ERK, ERK, p-AKT, and AKT were purchased from Cell Signaling Technology. Antibodies against MVP and GAPDH were purchased from Santa Cruz Biotechnology. Quantification was performed with Image J.
Capase 3
Manufactured by Cell Signaling Technology
Sourced in United States
Caspase 3 is a key executioner caspase that plays a central role in the execution-phase of cell apoptosis (programmed cell death). It is responsible for the proteolytic cleavage of many key cellular proteins, leading to the characteristic morphological and biochemical changes observed in apoptotic cells.
Automatically generated - may contain errors
Lab products found in correlation
P stat3, by Cell Signaling Technology (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
Lmnb1, by Cell Signaling Technology (1 mentions)
P jak, by Cell Signaling Technology (1 mentions)
P raf, by Cell Signaling Technology (1 mentions)
Cyclin d, by Cell Signaling Technology (1 mentions)
P mek, by Cell Signaling Technology (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
2 7 dichlorofluorescein diacetate, by Merck Group (1 mentions)
β actin monoclonal antibody, by Cell Signaling Technology (1 mentions)
Terminal deoxynucleotidyl transferase dutp nick end labeling tunel assay kit, by Beyotime (1 mentions)
Branched pei, by Merck Group (1 mentions)
T akt, by Cell Signaling Technology (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Agno3, by Merck Group (1 mentions)
Vitamin c, by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
8 protocols using capase 3
1
Comprehensive Protein Analysis via Western Blot
2
Evaluating Cytotoxicity of Nanoparticles
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The HepG2 cells were obtained from American Type Culture Collection (ATCC®, CCL-136™). LO2 cells (normal human liver cells line) were provided from Cell Bank of the Chinese Academy of Sciences (Shanghai, People’s Republic of China). Dulbecco’s Modified Eagle’s Medium and fetal bovine serum purchased from Gibco were used for cell culture. PTX, AgNO3, vitamin C, branched PEI, propidium iodide (PI), 2′,7′-dichlorofluorescein diacetate, 4′6-diamidino-2-phenylindole (DAPI), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were all obtained from Sigma. Capase-3, poly(ADP-ribose) polymerase (PARP), H2X, P-p53, P53, TAKT, T-p38, and β-actin monoclonal antibody were purchased from Cell Signaling Technology. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay kit, Annexin-V-FLUOS staining kit, caspase-3 activity assay kit, and BCA protein assay kit were acquired from Beyotime Institute of Biotechnology (Shanghai, People’s Republic of China).
3
Western Blot Analysis of Signaling Proteins
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Cells receiving various treatments and collected at various time points were subjected to western blot analysis, as previously described [35 (link),37 (link)]. Primary antibodies against IL-24 (1:2000; Introgen Therapeutics, Houston, TX, USA), GLI1, GLI2, PTCH2, SMO, γ-H2AX, PARP, Capase3, pATMS1981, ATM, pCHK2T68, CHK2, MRE11 (1:1000; Cell Signaling Technology Inc.), PTCH1 (Abcam, Cambridge, MA, USA), RAD50 (Santa Cruz Biotechnology, Dallas, TX, USA), and beta-actin (1:2000; Sigma Chemicals, St. Louis, MO, USA) were purchased and used as recommended by the manufacturers. Proteins were detected using the appropriate secondary antibodies (Santa Cruz Biotechnology, Inc., and Jackson Immuno Research Laboratories, Inc., West Grove, PA, USA) and an enhanced chemiluminescence kit (Thermo Scientific). Protein levels were detected using a chemiluminescence imaging system (Syngene, Frederick, MD, USA) and quantified using GelQuant software (V1.7.8, University of California- San Francisco, CA, USA).
4
Immunohistochemical Analysis of Tumor Markers
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At the end of the experiment, tumors from different groups were collected and washed with PBS, fixed in 4% formaldehyde and embedded in paraffin. Embedded tissues were cut into 4 mm slices for immunohistochemical analyses of Capase 3 (1:50 dilution in 5% bovine serum albumin (BSA); Cell Signaling Technology, Danvers, Massachusetts, USA), Ki67 (1:200 dilution in 5% BSA; ABclonal, Woburn, MA, USA), and CD34 (1:200 dilution in 5% BSA; Abnova, Taipei, Taiwan). Tissue slices were visualized under an optical microscope (Nikon Eclipse 80i, Tokyo, Japan) and subsequently analyzed with Image J2x software (National Institutes of Health, Bethesda, Maryland, USA). The ratio of cells staining positive for Capase 3 and Ki67 in each image was determined as a ratio of apoptotic or proliferative cell number to total tumor cell number. For CD34 staining, microvessel density (MVD) was quantified by counting the number of capillaries per microscopic field within five random fields in the hot spot per slide at 400x magnification.
5
Immunoblotting and Co-immunoprecipitation Techniques
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Immunoblotting and co-immunoprecipitations were performed as described [48 (link)]. Antiserum against FOXO (C29H4) and anti-Flag M2 (F3165) was purchased from Cell signaling Technology and Sigma for immunoblot analysis. E2F1 antibody (C20) was purchased from Sigma. Anti-HA antibody was purchased from Roche (3 F10). Anti-p53 (sc-6243, Santa Cruz), p-FOXO1 (9461S, Cell Signaling Technology), p-Akt (sc-7985-R, Santa Cruz), Capase-3 (Cell Signaling Technology, Cat# 9661), Ki-67 (SP6) (Biocare Medical, Cat# CRM325), and TUNEL (EMD Millipore, Cat# S7101) were used for IHC. IHC detection was through primary antibodies listed in Additional file 1 : Table S2, with Vector biotinylated secondary (1:250), tertiary was streptavidin-horseradish peroxidase (Covance #SIG-32000) and chromagen 3,3′-diaminobenzidine substrate (Covance #SIG-31043).
6
Molecular Mechanisms in MSC and SCC-25 Cells
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MSC, SCC-25, sorted SCC-25 and fused MSC/SCC cells were seeded at 5 × 105 cells/dish in 100-mm cell culture dishes. Cell lysates (20 μg) were electrophoresed and transferred to polyvinylidene fluoride (PVDF) membranes for probing for p-AKT (Cell Signaling Technology, Danvers, MA USA), p-STAT3 (Cell Signaling Technology), β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), E-cadherin (Cell Signaling Technology), vimentin (Santa Cruz Biotechnology), PARP1 (Santa Cruz Biotechnology), capase-3 (Cell Signaling Technology), and Bcl2 (Cell Signaling Technology). Western blots were visualized using alkaline phosphatase-conjugated secondary antibodies (Sigma-Aldrich, St Louis MO, USA).
7
Histological Analysis of Aortic Root
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Serial sections (6 μm thick) of the aortic root (3–5 sections per mouse) were stained with hematoxylin and eosin (HE), Oil Red O (ORO), or Masson’s trichrome (MASSON) and the microscopic images were then collected. Quantitative immunostaining was performed using primary antibodies for capase-3 (1:200, Cell Signaling Technology [CST], Danvers, MA, USA), Mac-3 (1:200, Abcam, USA), MOMA-2 (1:200, Abcam, USA), or α-smooth muscle actin (α-SMA, 1:100, Abcam, USA). Fluorescently labeled secondary antibodies were used for detection. The quantification of colocalized signals and the percentage of positive area in the images were performed using NIS Elements AR Imaging Software 4.10 (Nikon) or ImageJ 1.41 software (Image Processing and Analysis in Java; National Institutes of Health, Bethesda, MD, USA).
8
Mitochondrial Respiration and Signaling Modulation
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Oligomycin, rotenone, and cyanide p‐trifluoromethoxyphenyl‐hydrazone (FCCP) were obtained from Seahorse Biosciences; recombinant IL‐22 was provided by Novoprotein (China); cisplatin (Cisp.), MitoTracker Green, LY294002, 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethylbenzimidazolylcarbocyanine iodide (JC‐1), and compound C were obtained from Beyotime Biotechnology (China); antibodies targeting Glut1, β‐Actin, p‐AMPKα, AMPK, and GAPDH were purchased from Abcam; antibodies for STAT3, p‐STAT3 (Y705), AKT, p‐AKT (S473), PFKFB3, and Capase‐3 were purchased from Cell Signaling Technology; rapamycin, palmitic acid (Palm.), Z‐VAD‐FMK, and glucose were obtained from Sigma‐Aldrich; MitoSOX, Hoechst33342, MitoTracker Red, and PKH26 were obtained from Invitrogen.
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