These analyses were performed for MAFbx/Atrogin-1, MuRF1 and Gapdh mRNAs in plantaris muscles as described23 (link). Real-time PCR was performed using primers for MAFbox/Atrogin 1 and 18S rRNA from Applied Biosystems (Foster City, CA). Immunoblot analysis—Immunoblot analysis was performed as described6 (link) with the following primary antibodies: CuZnSOD (Abcam), MnSOD (Abcam), EcSOD (R&D, Minneapolis), peroxisome proliferator-activated receptor γ coactivator 1-α (PGC- 1α) (Chemicon/Millipore), cytochrome oxidase IV (COX IV) (Invitrogen), cytochrome C (Cyt C) (Cell Signaling, Danvers), malondialdehyde (MDA) (Academy Biomedical Company, Inc., Houston) and 4-hydroxynonenal (4-HNE) (ab48506, Abcam). Hybridoma for antibodies against MHC I (BA-F8), MHC IIa (SC-71) and MHC IIb (BF-F3) were purchased from German Collection of Microorganisms and Cell Cultures. All the bands for analysis have been validated previously3 (link), 6 (link), 24 (link). OxyBlot Protein Oxidative Detection Kit (Millipore) was used for immunblot detection of carbonylated proteins. Immunoblots were analyzed by Odyssey Infrared Imaging System (LI-COR Biosciences, NE) and quantified by Scion Image software.
Cytochrome c cyt c
Manufactured by Cell Signaling Technology
Sourced in United Kingdom, United States
Cytochrome c (cyt c) is a heme-containing protein that functions as an electron carrier in the mitochondrial electron transport chain. It plays a crucial role in cellular respiration and energy production.
Automatically generated - may contain errors
Lab products found in correlation
Caspase 3, by Cell Signaling Technology (5 mentions)
Cleaved caspase 3, by Cell Signaling Technology (3 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Gapdh monoclonal antibody, by Cell Signaling Technology (2 mentions)
Cell lysis buffer, by Cell Signaling Technology (2 mentions)
Bax 6a7, by Santa Cruz Biotechnology (2 mentions)
Coxiv polyclonal antibody, by Beyotime (2 mentions)
Phototope hrp chemiluminescent substrate system, by Cell Signaling Technology (2 mentions)
Caspase 9, by Cell Signaling Technology (2 mentions)
Anti mcl 1, by Cell Signaling Technology (2 mentions)
Bcl xl, by Cell Signaling Technology (2 mentions)
Bcl 2, by Santa Cruz Biotechnology (2 mentions)
Ab48506, by Abcam (1 mentions)
Ecsod, by R&D Systems (1 mentions)
Bf f3, by Leibniz Institute DSMZ (1 mentions)
Mnsod, by Abcam (1 mentions)
Cuznsod, by Abcam (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
B cell lymphoma 2 bcl 2, by Cell Signaling Technology (1 mentions)
9 protocols using cytochrome c cyt c
1
Oxidative Stress Signaling in Muscle Atrophy
2
Protein Expression Analysis by Western Blotting
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Western blotting was performed as previously described (Wang et al., 2015 (link)). Briefly, cell protein extracts (15–20 μg) were separated by 10–15% SDS-PAGE and transferred to PVDF membranes. Proteins were detected with the following antibodies: SIRT3 (1:1000, Cell Signaling Technology, Boston, MA, USA), MnSOD (1:1000, Proteintech, Chicago, IL, USA), cyclophilin D (CypD; 1:1000, Abcam, Cambridge, UK), cytochrome C (Cyt C; 1:1000, Cell Signaling Technology), cleaved caspase-3 (1:1000, Cell Signaling Technology), Bax (1:1000, Cell Signaling Technology, Boston, MA, USA), B-cell lymphoma 2 (Bcl-2; 1:1000, Cell Signaling Technology, Boston, MA, USA), COX-IV (1:1000, Abcam, Cambridge, MA, USA) and GAPDH (1:2000, Proteintech, Chicago, IL, USA).
3
Endothelial Cell Medium Metabolic Regulation
4
Exploring BM-1197's Impact on Apoptosis Signaling
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OCI-ly8 cells were treated with different concentrations of BM-1197 (0, 0.25, 0.5, 1, 2 μM) for 24 h or with 2 μM BM-1197 for 0, 1, 3, 6, 12 and 24 h. After that, cells were collected and washed twice with cold PBS and lysed in 1 × cell lysis buffer, which was diluted from 10 × cell lysis buffer (Cell Signaling Technology). Then, 1x proteinase inhibitor cocktail was added in the lysis buffer. Lysates were centrifuged at 12000 g at 4 °C for 20 min. Supernatants were collected and stored at − 80 °C until used. The protein concentration of the supernatants was determined using BCA protein assay reagents. The relevant primary antibodies used included: Bcl-2, Bax (6A7) and PARP-1 polyclonal antibodies purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and anti-Mcl-1, PUMA, Bcl-xl, caspase 3, Caspase 9, cytochrome c (cyt c), and GAPDH monoclonal antibody purchased from Cell Signaling Technology (Danvers, MA). The COXIV polyclonal antibody was purchased from Biyun Tian Biotechnology Research Institute. Mouse and rabbit secondary antibodies were from Cell Signaling Technology (Danvers, MA). Antigen-antibody complexes were detected using the PhototopeTM-HRP Chemiluminescent Substrate System (Cell Signaling Technology) per the manufacturer’s instructions.
5
Mitochondrial Dynamics and Apoptosis Signaling
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Liver tissues or cells were lysed in RIPA buffer. Lysates were centrifuged (14,000 x g for 10 min) and the supernatant was collected. The proteins were size-separated by 10% SDS-PAGE, transferred to PVDF membranes, and blocked with 5% non-fat dry milk before being incubated with primary antibodies: mitofusin-1 (MFN1) (Proteintech, Chicago, IL), dynamic-related protein-1 (DRP1) (Proteintech), fission-1 (FIS1) (Santa Cruz Biotechnology, Santa Cruz, CA), apoptosis signal-regulating kinase 1 (ASK1) (Cell Signaling Technology, Danvers, MA), phosphorated-c-Jun N-terminal Kinase (p-JNK) (Cell Signaling Technology), phosphorated-c-Jun (p-c-Jun) (Cell Signaling Technology), apoptosis protease activating factor-1 (Apaf-1) (Proteintech), cytochrome c (Cyt c) (Cell Signaling Technology), caspase 3 (Cell Signaling Technology), cleaved caspase 3 (Cell Signaling Technology), PARP (Cell Signaling Technology), cleaved PARP (Cell Signaling Technology), apoptosis-inducing factor (AIF) (Cell Signaling Technology), and endonuclease G (EndoG) (Cell Signaling Technology). Secondary antibodies were anti-mouse IgG and anti-rabbit IgG (1:5000 dilutions, Cell Signaling Technology). The membrane was visualized using the ECL-Plus chemiluminescence reagent (GE Healthcare, Buckinghamshire, UK).
6
Exploring BM-1197's Impact on Apoptosis Signaling
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OCI-ly8 cells were treated with different concentrations of BM-1197 (0, 0.25, 0.5, 1, 2 μM) for 24 h or with 2 μM BM-1197 for 0, 1, 3, 6, 12 and 24 h. After that, cells were collected and washed twice with cold PBS and lysed in 1 × cell lysis buffer, which was diluted from 10 × cell lysis buffer (Cell Signaling Technology). Then, 1x proteinase inhibitor cocktail was added in the lysis buffer. Lysates were centrifuged at 12000 g at 4 °C for 20 min. Supernatants were collected and stored at − 80 °C until used. The protein concentration of the supernatants was determined using BCA protein assay reagents. The relevant primary antibodies used included: Bcl-2, Bax (6A7) and PARP-1 polyclonal antibodies purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and anti-Mcl-1, PUMA, Bcl-xl, caspase 3, Caspase 9, cytochrome c (cyt c), and GAPDH monoclonal antibody purchased from Cell Signaling Technology (Danvers, MA). The COXIV polyclonal antibody was purchased from Biyun Tian Biotechnology Research Institute. Mouse and rabbit secondary antibodies were from Cell Signaling Technology (Danvers, MA). Antigen-antibody complexes were detected using the PhototopeTM-HRP Chemiluminescent Substrate System (Cell Signaling Technology) per the manufacturer’s instructions.
7
Mitophagy Signaling Pathway Analysis
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The protein samples were separated on 10% SDS‐PAGE gel, and the fractionated proteins were transferred to polyvinylidene fluoride membranes. After blocking the nonspecific binding sites with 5% BSA and 0.1% Tween‐20 in Tris buffer saline for 1 hour, the membranes were incubated with primary antibodies against LC3 (diluted 1:1000; Abcam), P62 (diluted 1:1000; Abcam), PINK1 (diluted 1:1000; Abcam), Parkin (diluted 1:1000; Abcam), Cytochrome C (Cytc, diluted 1:1000; Cell Signaling Technologies) GAPDH (diluted 1:1000; Cell Signaling Technologies), and VDAC (diluted 1:1000; Cell Signaling Technologies).The protein bands were detected using an enhanced chemical luminescence system (Cell Signaling Technologies). GAPDH was used for cytosolic protein loading control, and VDAC was used for mitochondrial protein control.
8
FGF21 Regulation of Mitochondrial Function
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FGF21 was purchased from PeproTech. The miR‐130 agomir was obtained from RiboBio. The primary antibodies used in this study included PPARγ (Abcam, #ab178860), α‐SMA (Abcam, #ab5694), bcl‐2 (Abcam, #ab59348), proliferating cell nuclear antigen (PCNA; Cell Signalling Technology, #13110), β‐actin (Cell Signalling Technology, #4970), Bax (Cell Signalling Technology, #14796), caspase‐3 (Cell Signalling Technology, #9665), cleaved caspase‐3 (Cell Signalling Technology, #9665), apoptosis‐inducing factor (AIF; Cell Signalling Technology, #5318), cytochrome c (Cyt C; Cell Signalling Technology, #11940), cyclin‐dependent kinase 1 (CDK1; Affinity Biosciences, DF6024), cyclin D1 (Affinity Biosciences, AF0931) and COX IV (Affinity Biosciences, AF5468). The PPARγ agonist pioglitazone (Pio) was obtained from Selleck (Houston, TX, USA). Donkey anti‐mouse IgG H&L (Alexa Fluor 488; lot no. ab150105) antibodies were purchased from Abcam. Horseradish peroxidase (HRP)‐conjugated goat anti‐rabbit IgG antibody (lot no. BL003A) was purchased from Biosharp. Tissue Mitochondria Isolation Kit (#C3606) and Cell Mitochondria Isolation Kit (#C3601) were purchased from Beyotime. The One‐Step Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) Apoptosis Assay Kit (#C1089) was purchased from Beyotime.
9
Molecular Profiling of Cell Apoptosis
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Monoclonal antibodies specific of Caspase-3 (cleaved Caspase-3), Caspase-9 (cleaved Caspase-9), Bcl-2, myeloid cell leukemia-1 (Mcl-1), Bax, Bim, Cytochrome C (Cyt-C), and STAT3 were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). Caspase-3 and Caspase-9 activity assay kits were ordered from Abcam (Cambridge, MA, USA). Lipofectamine 3000 reagent was purchased from Life Technologies (Carlsbad, CA, USA). SiSTAT3 (#1,2,3) and all qRT-PCR primers including Bcl-2, Mcl-1, Bax, Bim, STAT3 and GAPDH were purchased from Ribobio (Guangzhou, Guangdong, China). All primer sequences and siSTAT3 target sequences were shown in Additional file 1 : Table S1. NSCLC cells A549 were obtained from the Cell Line Bank at the Laboratory Animal Center of Sun Yat-sen University (Guangzhou, China) and PC9 were obtained from the Chinese Academy of Sciences Cell Bank of Type Culture Collection (Shanghai, China). All cells were grown at 37 °C, in a humidified 5% CO2 and 95% air and cultured in RPMI-1640 medium (Life Technologies, Carlsbad, CA, USA) containing 10% FBS (Gibco, USA) and 0.5% penicillin–streptomycin sulfate (Invitrogen, Carlsbad, CA, USA). Cells were counted using the automated cell counter star (Invitrogen, Carlsbad, CA, USA).
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