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. 2023 May 13;14(5):325. doi: 10.1038/s41419-023-05846-5

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — A The calcium channels in the crude mitochondrial fraction isolated from the hippocampi of male WT rats were measured by western blotting. Total total homogenate, Mc crude mitochondria. The molecular weight of each protein is provided on the right. The stray band is indicated by an asterisk. B Diagram of the two calcium flow paths into isolated crude mitochondria. The arrows indicate the direction of calcium flux. The red box indicates calcium flow through the MAM. The green box indicates calcium flow through the OMM_cyto. Calcium flowing into the mitochondrial matrix through the MAM needs to pass through three layers, including the ER membrane, OMM and inner mitochondrial membrane (IMM), while calcium flowing through the OMM_cyto needs to cross only two layers, the OMM and IMM. A higher concentration of calcium should flow through the MAM than through the OMM_cyto because the calcium concentration in the ER lumen is usually 1000 times higher than that in the cytoplasm [28]. C Representative fluorescence traces showing mitochondrial calcium uptake after 8 μM calcium stimulation. Ten milligrams of isolated crude hippocampal mitochondria from 3-month-old male rats were loaded with Fluo 4-AM and used for the assay. The fluorescence was measured 6 times every 30 s before and after stimulation buffer was added. The average mitochondrial fluorescence count before stimulation was used as an internal reference (F₀). The fluorescence fold change relative to F₀ (F₁/F₀) was used for normalization to assess calcium uptake. The F₁/F₀ value is plotted on the y-axis, and the detection time is plotted on the x-axis. The red arrow indicates the addition of 8 μM CaCl₂ buffer for stimulation. D Three independent experiments including at least five rats per genotype were performed as described in (C). The average F₁/F₀ values after 8 μM calcium stimulation of hippocampal mitochondria from individual rats are indicated by dots. The mean ± SEM for each genotype is presented. One-way ANOVA was used for statistical analysis, and differences are indicated by asterisks. **p < 0.01 and ***p < 0.001 indicate significant differences, and n.s. indicates no significance. E, F Similar experiments were performed with 16 μM calcium stimulation. Representative fluorescence traces are shown in (E), and quantitative data for the four genotypes are presented in (F). The F₁/F₀ values of individual rats are indicated by dots. The mean ± SEM for each genotype is presented. One-way ANOVA was used for statistical analysis, and differences are indicated by asterisks. *p < 0.05 and ***p < 0.001 indicate significant differences.