Fig 1: ADSC graft characteristics. (A, B) Western blotting of mitochondrial ANT-1 and cytosolic ATP concentration in Groups ADSC and Sham at two and four weeks after ADSC transplantation. The blotting figure was cropped for clarity and full-length blots are presented in S1 Fig. (C) Cytokine secretion from ADSC graft. ANOVA and Student's t-test were used to evaluate significant differences, with *P < 0.05 versus Group Sham and †P < 0.05 versus Group Wild.
Fig 2: H2O2-induced programmed necrosis is inhibited by 4-OI in osteoblasts.Human osteoblastic OB-6 cells (a–c) or the primary murine osteoblasts (d, e) were pretreated for 2 h with 4-OI (10/25 µM) or vehicle control (“Veh”), followed by H2O2 (400 µM) stimulation. Cells were further cultured for applied time periods; mitochondrial CyPD-ANT1-p53 association and their expression are shown (a); mitochondrial depolarization was tested by JC-1 green monomer fluorescence (b, d), and cell necrosis examined by quantifying medium LDH release (c, e). Expression of the listed proteins was quantified and normalized to the loading control (a). Quantified values were mean ± standard deviation (SD, n = 5). “C” stands for the untreated control cells. *P < 0.05 vs. “C” cells. #P < 0.05 vs. cells with H2O2 stimulation but “Veh” pretreatment. Experiments were repeated three times, with similar results obtained. Bar = 100 µm (b).
Fig 3: SS-31 interacts with ANT1 and stabilizes the ATP synthasome in the old mouse heart mitochondria.(A, B) Biotin-SS-31 pulldown shows the association of biotin-SS-31 to ANT1. Free SS-31 competes with this interaction, while Bongkrekic Acid (BKA) and carboxyatractyloside (CAT) inhibit the interaction of biotin-SS-31 with ANT1. Panel A shows a representative Western blot. N = 6 mice in each group. One-way ANOVA with Fisher’s LSD test was applied. **p<0.01 vs Biotin control, #p<0.05, ##p<0.01 vs Biotin-SS31 pulldown. (C) Coomassie blue staining of isolated mitochondria in a native gel. (D) Left panel is the total protein loading control for the Native gel blot. (D, E) Native gel blotting shows that 10 µM SS-31 stabilizes the mitochondrial synthasome (Syn) in isolated mitochondria. The Syn is highlighted in the red box. The Syn and ATPase Dimer (D) and Monomer (M) were labeled using anti-ATP5A. N = 8 mice in each group. One-way ANOVA with Fisher’s LSD test was applied. *p<0.05 vs young, #p<0.05 vs old.
Fig 4: ANT1 inhibitors restore resistance to proton leak of mitochondria in old rat cardiomyocytes.ANT1 inhibitors 10 µM Bongkrekic Acid (BKA) and 20 µM carboxyatractyloside (CAT), but not 50 µM Genipin (UCP2 inhibitor) or 1 µM oligomycin A (OA, ATPase inhibitor), protected the mitochondrial matrix from decreased pH after exposure to external pH 5.3 (N = 4–19 rats in each group) (A) and reduced the rate of 488/405 decline after exposure to pH 6.9 (N = 4–10 rats in each group) (B). BKA, CAT, Genipin, or OA were added immediately after the mitochondria permeabilization. One-way ANOVA with Fisher’s LSD test was applied. *p<0.05, **p<0.01 vs Young, #p<0.05, ##p<0.01 vs Old.
Fig 5: RBFOX2 regulates tandem APA and expression of essential mitochondrial gene Slc25a4(A) Slc25a4 3'UTR lengthening mediated via tandem APA in RBFOX2-depleted H9c2 myoblasts identified by PAC-seq. The number of reads mapped to Slc25a4 transcripts is shown on the right side. See also Figure S5.(B) Nanopore sequencing analysis of full-length Slc25a4 transcripts with different 3'UTR lengths in control versus RBFOX2 KD myoblasts. See also Figure S5.(C) RT-PCR analysis of endogenous Slc25a4 dPAS/total mRNA ratio in RBFOX2 KD myoblasts ectopically expressing GFP or GFP-RBFOX2. n = 8, ****p < 0.0001 (three independent experiments). See Table S2 for primer information.(D) WB analysis of ANT1 protein (Slc25a4) in control versus RBFOX2 KD myoblasts determined by Bio-Rad ChemiDoc Imager. Even protein loading was monitored by imaging stain-free gels. ANT1 protein levels were normalized to loading control, and fold change in ANT1 protein levels was quantified using Bio-Rad ChemiDoc software. n = 4, ***p = 0.0002 (three independent experiments).
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