Fig 1: RAP1GDS1/Miro1 and mitochondrial functional change mouse brain aging.a Western blot analysis of RAP1GDS1 (antibody from Abcam #ab188020) and Miro1 change during aging in the brain tissue of C57 mice. β-actin is the protein loading control. Uncropped blots are shown in Supplementary Fig. 8. With the 2 month-old sample set as 1. N = 6. One-way ANOVA with Tukey’s post hoc test. 2vs21, *P = 0.023; 2vs27, ****P < 0.0001; 16vs27, **P = 0.0035. b Co-Immunoprecipitation of RAP1GDS1 and Miro1 during aging. IP with RAP1GDS1 antibody (Santa #sc39003) to pull down Miro1. Total protein input is shown as the protein loading control. Uncropped blots are shown in Supplementary Fig. 8. The ratio of Miro1/RAP1GDS1 from the 2 month-old sample is set as 1, and the relative level of other samples are shown. N = 3. One-way ANOVA with Tukey’s post hoc test. **P = 0.0056. c Representative mitochondrial morphology stained by VDAC1 in neurons of prefrontal cortex. Scale bar, 5 μm. Mitochondrial size changes in brain neurons during aging. 2 month-old sample is set as 1. N = 10. One-way ANOVA with Tukey’s post hoc test. ****P < 0.0001. d Brain mitochondrial calcium concentration ([Ca2+]mito.) change during aging. The intensity of Rhod2-AM indicates [Ca2+]mito level. Each mouse brain quantified 20 neurons, the Rhod-2AM intensity from 2 month-old sample was set as 1, and the ratios of others are shown. N = 4. One-way ANOVA with Tukey’s post hoc test. ****P < 0.0001, ****P < 0.0001. e Cytosolic calcium ([Ca2+]cyto) change stained by BAPTA-1 AM in young (2 month-old) and senescent (27 month-old) mice. The BAPTA-1 AM intensity of 2 month-old is set as 1. N = 6. Error bars are mean ± SE. Unpaired t-test. ****P < 0.0001.