In this study, sinularin was obtained from National Museum of Marine Biology & Aquarium (Pintung, Taiwan) and the chemical structure showed in Figure 1 B. The anti-β-actin antibody was obtained from Sigma-Aldrich (St. Louis, MO, USA), apoptosis antibody sample kit, pro-apoptosis bcl-2 family antibody sample kit, pro-survival Bcl-2 family antibody sample kit, phospho-pI3 kinase p85 (Tyr458)/P55 (Tyr799) antibody, phosopho-GSK3β (Ser9) (D3A4) rabbit antibody, PI3 kinase p110α antibody were obtained from Cell Signaling (Danvers, MA, USA), anti-cytochrome C, CD95/Fas, AIF, phospho-Akt1, Akt1, mTOR/FRAP and phosopho-mTOR (Phospho-Ser2481) antibody were obtained from Epitomics (Bellerica, MA, USA). Goat anti-rabbit and horseradish peroxidase conjugated IgG was obtained for Millipore (Bellerica, MA, USA).
Anti cytochrome c
Manufactured by Abcam
Sourced in United Kingdom, United States, China
Anti-cytochrome c is a primary antibody that specifically recognizes cytochrome c, a mitochondrial protein involved in the electron transport chain. This antibody can be used in various applications, such as Western blotting, immunohistochemistry, and flow cytometry, to detect and quantify the presence of cytochrome c in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti bax, by Abcam (10 mentions)
Anti bcl 2, by Abcam (8 mentions)
Anti caspase 3, by Abcam (5 mentions)
Anti caspase 9, by Abcam (5 mentions)
Anti cleaved caspase 3, by Abcam (5 mentions)
Anti β actin, by Abcam (4 mentions)
Lsm 700 meta, by Zeiss (4 mentions)
Lsm image browser, by Zeiss (4 mentions)
Anti cleaved caspase 9, by Abcam (4 mentions)
Anti β actin, by Merck Group (4 mentions)
Anti bcl 2, by Santa Cruz Biotechnology (3 mentions)
Anti bcl xl, by Abcam (3 mentions)
Anti p62, by Abcam (3 mentions)
Chemidoc xr, by Bio-Rad (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Chemidoc machine, by Bio-Rad (2 mentions)
Anti β actin, by Santa Cruz Biotechnology (2 mentions)
Formaldehyde solution, by R&D Systems (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
45 protocols using anti cytochrome c
1
Anticancer Effects of Sinularin
2
Western Blot Analysis of Cellular Markers
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Western blot analysis was performed, as previously described (14 (link)). Briefly, the primary antibodies used to probe the membranes included anti-UCP2 (1:500, cat. no. ab67241; Abcam, Cambridge, UK), anti-cytochrome c (1:200, cat. no. 1896-1; Epitomics, Burlingame, CA, USA), anti-caspase-3 (1:5,000, cat. no. ab32351; Abcam) and anti-β-actin (1:1,000, cat. no. A1978; Sigma-Aldrich, St. Louis, MO, USA). Experiments were repeated in triplicate.
3
Investigating Apoptosis Pathway in GC-2spd Cells
4
Lung Cancer Cell Line A549 Culture
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Human non-small cell lung cancer cell line A549 (CCL-185) was obtained from Chinese Academy of Sciences Cell Bank of Type Culture Collection (CBTCCCAS) and cultivated in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 5% fetal bovine serum, 2 mM L-glutamine, 100 U/L penicillin and 0.1 mg/ml streptomycin (all from Life technology, Grand Island, NY) and maintained in a humidified incubator with 5% CO2 at 37°C.
Antibodies used in this study were: anti-LC3 (Thermo Scientific, Waltham, MA, PAI-16930, 1:500 dilution), anti-p62/SQSTM1 (Epitomics, Burlingame, CA, #3340-1, 1:3000 dilution), anti-HSP60 (Epitomics, #1724-1, 1:10000 dilution), anti-caspase-3 (Cell Signaling Technology, #9662, 1:1000 dilution), anti-caspase-9 (Cell Signaling Technology, #9502, 1:1000 dilution), anti-cytochrome c (Epitomics, #2119-1, 1:1000 dilution), anti-HSP60 (Epitomics, #1724-1, 1:3000 dilution), anti-GAPDH (Bioworld, Minneapolis, MN, MB001, 1:5000 dilution) and HRP-conjugated secondary antibodies (Multisciences, Hangzhou, China, GAR007 and GAM007, 1:5000 dilution).
The following reagents, 3-Methyladenine (3-MA, #M9281), chloroquine (CQ, #C6628), z-VAD-fmk (#V116) and trypan blue (#T6146) were all obtained from Sigma-Aldrich (Saint Louis, MO).
Antibodies used in this study were: anti-LC3 (Thermo Scientific, Waltham, MA, PAI-16930, 1:500 dilution), anti-p62/SQSTM1 (Epitomics, Burlingame, CA, #3340-1, 1:3000 dilution), anti-HSP60 (Epitomics, #1724-1, 1:10000 dilution), anti-caspase-3 (Cell Signaling Technology, #9662, 1:1000 dilution), anti-caspase-9 (Cell Signaling Technology, #9502, 1:1000 dilution), anti-cytochrome c (Epitomics, #2119-1, 1:1000 dilution), anti-HSP60 (Epitomics, #1724-1, 1:3000 dilution), anti-GAPDH (Bioworld, Minneapolis, MN, MB001, 1:5000 dilution) and HRP-conjugated secondary antibodies (Multisciences, Hangzhou, China, GAR007 and GAM007, 1:5000 dilution).
The following reagents, 3-Methyladenine (3-MA, #M9281), chloroquine (CQ, #C6628), z-VAD-fmk (#V116) and trypan blue (#T6146) were all obtained from Sigma-Aldrich (Saint Louis, MO).
5
Western Blot Analysis of Cardiac Proteins
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Total proteins from H9c2 cells and working heart tissues were extracted using RIPA lysis buffer, separated by electrophoresis on an SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MS, USA). The membrane was blocked with 5% nonfat milk in Tris-buffered saline/Tween 20 (TBST) and was incubated overnight with the following primary antibodies: anti-ox-CaMKIIδ (Met281/282) (1 : 1000, GeneTex), anti-p-CaMKIIδ (Thr286) (1 : 1000, Abcam), anti-CaMKIIα (1 : 10000, Abcam), anti-CaMKIIβ (1 : 500, Abcam), anti-CaMKIIδ (1 : 1000, Abcam), anti-CaMKIIγ (1 : 500, Abcam), anti-p-JNK1/2 (T183/Y185) (1 : 1000, CST), anti-JNK1/2 (1 : 1000, CST), anti-p-NF-κB p65 (Ser536) (1 : 1000, Abcam), anti-NF-κB p65 (1 : 1000, Abcam), anti-Bcl-2 (1 : 500, Abcam), anti-Bax (1 : 500, Abcam), anti-cytochrome c (1 : 1000, Abcam), and anti-GAPDH (1 : 1000, ZSGB-Bio, Beijing, China). The membrane was then washed and probed with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse antibody (1 : 8000, ZSGB-Bio) to enhance the chemiluminescence that was detected using a Fusion-FX6 imaging system (Vilber Lourmat, Marne-la-Valle, France).
6
Evaluating Cellular Stress Markers in Breast Cancer Cell Lines
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MDA-MB-468 cells and MCF-7 were treated with the indicated concentrations of ChPL for 24 h, and Western blot analysis was performed according to the previously published procedure (Kawiak et al., 2012b (link)). The preparation of cytosolic and mitochondrial fractions for cytochrome c release evaluation was performed as previously described (Kawiak et al., 2012b (link)). The following specific primary antibodies were used: anti-β-actin (1:1,000) (Cell Signaling, Danvers, MA, USA), anti-Bcl-2, anti-Bak, anti-Bax, and anti-Mcl-1 (1:250) (Santa Cruz, Heidelberg, Germany), anti-ERK1/2, anti-MEK1/2, anti-p-ERK1/2, and anti-p-MEK1/2 (1:1,000) (Cell Signaling), anti-cytochrome c (1:5,000) (Abcam, UK), and anti-HSP60 (1:1,000) (Cell Signaling). Membranes were incubated with primary antibodies overnight at 4°C after which a 1-h incubation with HRP-conjugated secondary antibodies (1:2000) (Cell Signaling) was carried out. Protein levels were determined by chemiluminescence (ChemiDoc; Bio-Rad, Waltham, MA, USA) with a HRP substrate (Thermo Scientific, MA, USA).
7
Apoptosis Regulation in Leukemia Cells
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KG-1 cells were plated in a 6-well plate at a density of 106 cells/mL before treatment with two increasing concentrations of ASEE for 24 h (38 and 76 μg/mL). Control cells were treated with RPMI media. Total proteins were extracted using the Q-proteome Mammalian Protein kit (Qiagen, Hilden, Germany) and quantified using the DC (Detergent Compatible) protein assay (Bio-Rad). Proteins were separated by SDS-PAGE; transferred to PVDF (Polyvinylidene fluoride) membranes which were blocked with 5% skimmed milk; and then incubated with primary antibodies anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA), anti-Cytochrome-c, anti-cleaved PARP-1, anti-Bax, anti-Bcl2, and anti-caspase-9 (Abcam, Cambridge, UK), anti-p53, and anti-caspase-8 (Elabscience, Houston, TX, USA) at the manufacturer’s recommended concentrations. After washing and incubation with a secondary antibody (Bio-Rad, Hercules, CA, USA), membranes were washed and image development was done using the Clarity™ Western ECL Substrate (Abcam, Cambridge, UK) on the ChemiDoc machine (BioRad, Hercules, CA, USA). Blot bands were quantified using the ImageJ computer program to calculate the relative expression of proteins.
8
Cytochrome c Immunofluorescence Staining
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The expression of cytochrome c was analyzed by immunofluorescence staining. Cells grown on glass-bottomed dishes (MatTek, Ashland, MA, USA) were fixed with 4% formaldehyde solution (R&D Systems, Abingdon, UK) for 10 min and permeabilized with 0.5% TritonX-100 in phosphate-buffered saline (PBS) for 10 min. Slides were air-dried, washed with PBS, and incubated with anti-cytochrome c (1:25; Abcam, Cambridge, UK) in 3% bovine serum albumin in PBS. Further details on the protocol can be found in our previous article [68 (link)]. Images were observed under a confocal microscope (LSM Meta 700; Zeiss, Oberkochen, Germany) and were analyzed using Zeiss LSM Image Browser, version 4.2.0121.
9
Mitochondrial Apoptosis Pathway Analysis
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Sample treatment was referred to our previous study [29 (link)]. Anti-Cytochrome C (1:5000, Abcam), anti-Cleaved Caspase-3 (1:5000, Abcam) and anti-Cleaved Caspase-9 (1:5000, Abcam) antibodies were used to detect the effects of TGN/SHp/TPP-MLT micelles on the mitochondrial apoptosis pathway. The cytosolic and mitochondrial fractions of PC-12 cells were isolated by standard differential centrifugations using a a mitochondrion isolation kit (Sigma-Aldrich, USA). Normalization was ensured by VDAC-1 and β‐actin, and the target bands were quantified by ImageJ software.
10
Cytochrome c Immunofluorescence Assay
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The level of cytochrome c was evaluated by the immunofluorescence staining assay. The primary antibody for the assay was anti-cytochrome c (1:25; Abcam, Cambridge, UK) incubated in 3% bovine serum albumin in PBS. Nuclei were stained with Hoechst 33342 (Life Technologies, Grand Island, NY, USA). Images for immunofluorescence were acquired using a confocal microscope (LSM Meta 700; Zeiss, Oberkochen, Germany) and were analyzed with Zeiss LSM Image Browser, version 4.2.0121.
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