Journal: Artificial cells, nanomedicine, and biotechnology

Article Title: Zinc oxide nanoparticles induce murine photoreceptor cell death via mitochondria-related signaling pathway.

doi: 10.1080/21691401.2018.1446018

Figure Lengend Snippet: Figure 5. Determinations of Bax, Bcl-2 and Caspase 3 expressions at mRNA and protein levels after exposure to different concentrations of ZnO nanoparticles for 6 h. (A), (B) and (C) were determined using quantitative PCR, (D), (E) and (F) were determined by ELISA. p < .05 and p < .01 versus relevant controls.

Article Snippet: The supernatant of each sample was collected and the content of cytochrome c released from cells was determined by a Mouse Cytochrome-c (Cyt-C) ELISA Kit (CSB-E08532m) according to the manufacturer’s instructions (CUSABIO, Biotech Co., Ltd., Wuhan, China).

Techniques: Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Molecules and Cells

Article Title: Purification and Characterization of Mitochondrial Mg 2+ -Independent Sphingomyelinase from Rat Brain

doi: 10.14348/molcells.2023.0074

Figure Lengend Snippet: (A) A scheme of the isolation process of mitochondria from rat brain. (B) Immunoblot for cytochrome c , Hsp60, SNAP25, nSMase1, and nSMase2 from each fraction in (A). (C) Magnesium-dependent (with 10 mM MgCl2) or independent (with 5 mM EDTA) SMase activities were measured with 20 μg of proteins in each fraction of (A) for 10 min. (D) SMase activity in mitochondrial and synaptosomal fractions isolated from rat brain. (Upper) Synaptosomal and mitochondrial fractions were analyzed by immunoblotting for SNAP25, Hsp60, cytochrome c , and nSMase2. (Lower) SMase activities were measured with 20 μg of proteins from the synaptosomal and mitochondrial fractions with 10 mM MgCl2 or 5 mM EDTA for 10 min. (E and F) Effects of GW4869 on SMase activities in the mitochondrial (iSMase enriched, E) and S1.3 (nSMases enriched, F) fractions. The fractions were pre-incubated with 0, 1, 5, and 10 μM of GW4869 in the presence of 5 mM EDTA or 10 mM MgCl2 for 10 min at 37°C. The SMase activities were then determined in the presence of 2.5 μM [ 14 C] SM (sphingomyelin) for 10 min at 37°C. All error bars represent mean ± SEM. All data are representative of three independent experiments. * P < 0.05, *** P < 0.001.

Article Snippet: Purified Mouse Anti-cytochrome c antibody was purchased from BD Biosciences (USA).

Techniques: Isolation, Western Blot, Activity Assay, Incubation

Journal: Molecules and Cells

Article Title: Purification and Characterization of Mitochondrial Mg 2+ -Independent Sphingomyelinase from Rat Brain

doi: 10.14348/molcells.2023.0074

Figure Lengend Snippet: (A) Mitochondria were sub-fractionated into outer membrane (OM), IMS, inner membrane (IM), and matrix (M). Equal amounts of each fraction were analyzed for mt-iSMase activity (A, lower) and immunoblotted with the following mitochondrial markers for IM, OM, IM, and M: cytochrome c , VDAC, COX IV, and Hsp60, respectively (A, upper). (B and C) Mitochondrial and synaptosomal fractions were immunoprecipitated with anti-Hsp60 and anti-c-Myc antibodies. (B) The immunoprecipitated supernatants and pellets were immunoblotted for Hsp60, SNAP25, and cytochrome c . (C) SMase activities in the supernatants and pellets were measured in the presence of 5 mM EDTA or 10 mM MgCl2. All error bars represent the mean ± SEM. All data represent three independent experiments. n.s., not significant; ** P < 0.01, *** P < 0.001. cpm, counts per minute; IP, immunoprecipitation.

Article Snippet: Purified Mouse Anti-cytochrome c antibody was purchased from BD Biosciences (USA).

Techniques: Membrane, Activity Assay, Immunoprecipitation