Cells or NK-EVs were lysed with NP40 Cell Lysis Buffer containing protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Cell lysates were incubated with Loading Dye (LI-COR) for 30 min under non-reducing conditions at 37 °C to detect CD63 as previously described [29 (link)] or with Loading Dye and 2-mercaptoethenol for 5 min at 95 °C to detect other proteins. Proteins were loaded onto NuPAGE gels (Invitrogen), transferred to membranes, and the membranes were incubated with 5% skim milk in TBS-T for 1 h at room temperature. The membranes were then incubated overnight at 4 °C with primary antibodies, anti-CD81, CD63 (Cosmo Bio, clone 12C4, 8A12, respectively), cytochrome c, granzyme B (Biolegend, clone 7H8.2C12, O94E6, respectively), β-actin, granzyme H (Cell Signaling Technology, catalog 4967, 18268, respectively), and α-tubulin (Santa Cruz, catalog sc-23948). Secondary antibodies, IRDye 680RD-conjugated anti-mouse IgG, or anti-rabbit IgG (LI-COR) were used at a 1:5000 dilution. The membranes were exposed to ODYSSEY CLx (LI-COR).
Cytochrome c
Manufactured by BioLegend
Sourced in United States, China
Cytochrome c is a small heme protein found in the mitochondria of eukaryotic cells. It is a vital component of the electron transport chain, a series of protein complexes involved in cellular respiration and the production of ATP. Cytochrome c plays a crucial role in the transfer of electrons between these protein complexes, facilitating the flow of energy within the cell.
Automatically generated - may contain errors
Lab products found in correlation
Anti rabbit igg, by LI COR (1 mentions)
Np40 cell lysis buffer, by Thermo Fisher Scientific (1 mentions)
Irdye 680 rd conjugated anti mouse igg, by LI COR (1 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Nupage gel, by Thermo Fisher Scientific (1 mentions)
Loading dye, by LI COR (1 mentions)
Anti cd81, by Cosmo Bio (1 mentions)
α tubulin, by Santa Cruz Biotechnology (1 mentions)
Granzyme b, by BioLegend (1 mentions)
Odyssey clx, by LI COR (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Oligomycin, by Merck Group (1 mentions)
Cox 4, by Cell Signaling Technology (1 mentions)
Anti mouse, by BioLegend (1 mentions)
Humulin r insulin, by Eli Lilly (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
Ccl20, by BioLegend (1 mentions)
Cytochrome c, by Thermo Fisher Scientific (1 mentions)
Mmp 3, by R&D Systems (1 mentions)
Cd81 sc 166 028, by Santa Cruz Biotechnology (1 mentions)
6 protocols using cytochrome c
1
Western Blot Analysis of EV Proteins
2
Mitochondrial Function and Lipid Metabolism
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All reagents were obtained from Sigma-Merck, except for oligomycin (cat. sc-201551), CCCP (cat. sc-202984) and palmitoyl-L-carnitine (cat. sc-203176), from Santa Cruz, and insulin (Humulin R), from Eli Lilly. VLCAD (cat. ab155138), CPT-1 (cat. ab128568) antibodies from Abcam; COX IV (cat. 4844) antibodies from Cell Signaling: cytochrome c (cat. 612504) from Biolegend, and anti-mouse (cat. 926–68070) and anti-rabbit IRDye (cat. 926–68071) were from LI-COR Biotechnology. Triglycerides (cat. 87) and cholesterol (cat. 76) kits were from Labtest. The NEFA kit (cat. 434.71795) was from Wako Chemicals.
3
Quantifying Inflammatory Mediators
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Chemokine, cytokine, cytochrome C, and MMP3 concentration was determined using enzyme-linked immunosorbent assay kits as per the manufacturers protocol; CCL20 (BioLegend), cytochrome C, IL-6, IL-8 (all Invitrogen), and MMP3 (R&D Systems).
4
EV Protein Characterization by Western Blot
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EV proteins (20 µg/lane) were resolved using sodium dodecyl sulphate-polyacrylamide gel electrophoresis, transferred onto polyvinylidene fluoride membranes, probed with each primary antibody, and incubated with a horseradish peroxidase-conjugated secondary antibody. The following primary antibodies were used: CD63 (D4I1X, 1:500) and CD9 (D801A, 1:500) from Cell Signaling Technology (Danvers, MA, USA), CD81 (sc166028, 1:500) from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), and cytochrome c (1:1,000) from BioLegend (San Diego, CA, USA).
5
Genistein-induced Apoptosis and Oxidative Stress
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Genistein (with purity >97%) was purchased from Shanghai Maclin Biochemical (Shanghai, China) and dissolved in 0.1% DMSO before use. A CCK-8 assay kit (Solarbio, Beijing, China), AnnexinV-FITC/PI apoptosis detection kit, and DAPI were purchased from BD Pharmingen (Franklin Lakes, NJ). 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) and dihydroethidium (DHE) probes were purchased from Beyotime Biotech (Nantong, China). B cell lymphoma-2 (Bcl-2, #244614), Bcl-2-associated x protein (Bax, #B323312), cytochrome C (#288539) and FOXO3a (#B217778) were purchased from Biolegend; GAPDH (#BM3896) was purchased from Boster Biological Technology; PUMA (#GR3239989-14) was purchased from Abcam; p-Akt (#DZE1819111) was purchased from Biotechne; and HPR-labeled goat anti-rabbit IgG (#A020) and HPR-labeled goat anti-mouse IgG (#8A0216) were purchased from the Beyotime Institute of Biotechnology (Nantong, China). MitoTracker Orange CMTMRos (M7510) was purchased from ThermoFisher Scientific (Waltham, MA).
6
Western Blot Analysis of Cellular Proteins
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Protein samples were resolved by SDS-PAGE and transferred to a PVDF membrane (Millipore). All primary antibodies were used at 1:1000 and secondary antibodies at 1:5000. The following antibodies were used: ACTB (Developmental Studies Hybridoma Bank, #JLA20-c); VDAC1 (Santa Cruz, sc-8828); cytochrome c (Biolegend, 612504); Caspase-3 (Cell Signaling, 9661); DIS3L1 (Abcam, ab89042); DIS3L2 (Novus, NBP1-84740); Caspase-9 (Cell Signaling, 9502); PABPC1 (Sigma, P6246); PNPT1 (Abcam, ab96176); anti-Mouse HRP (GE Healthcare, NA931V); anti-Rabbit HRP (GE Healthcare, NA934V); anti-Goat HRP (Santa Cruz, sc-2020) . Protein bands were visualized using a SuperSignal West Pico chemiluminescence ECL kit (Pierce).
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