Fig 1: SHP2 dephosphorylation of ANT1 at Tyr 191 is essential for mitochondrial homeostasis. a Flow cytometry analysis of mitochondrial membrane potential by JC-1 staining of HEK293T cells overexpressing Vector, SHP2-HA, SHP2-D61A-HA, or SHP2-C459S-HA plasmid and left untreated (medium) or treated with cccp (20 µM, 1 h). b,c HEK293T cells were transfected with pro-caspase-1, ASC, NLRP3, and SHP2-HA, SHP2-D61A-HA, or SHP2-C459S-HA plasmid, respectively, followed by ATP (5 mM, 1 h) treatment. b Flow cytometry analysis of caspase-1 activation. c Immunoblot analysis of caspase-1 activation. d Flow cytometry analysis of mitochondrial membrane potential by JC-1 staining of HEK293T cells overexpressing Vector, ANT1-myc, ANT1-Y191F-myc, or ANT1-Y195F-myc plasmid and left untreated (medium) or treated with cccp (20 µM, 1 h). e, f HEK293T cells were transfected with pro-caspase-1, ASC, NLRP3, and ANT1-myc, ANT1-Y191F-myc or ANT1-Y195F-myc plasmid respectively followed by ATP (5 mM, 1 h) treatment. e Flow cytometry analysis of caspase-1 activation. f Immunoblot analysis of caspase-1 activation. *P < 0.05, one-way ANOVA for multiple comparisons, NS represents no significance. Data are representative of three independent experiments (mean and SEM of three independent samples in a, b, d, e)
Fig 2: De Novo SLC25A4 Mutations Do Not Decrease Mitochondrial Protein Import(A) Western blot analysis of inner mitochondrial membrane proteins performed on skeletal muscle lysates from subject 2 (p.Arg80His).(B) Western blot analysis of inner mitochondrial membrane proteins performed on skeletal muscle samples from subject 5 (p.Arg235Gly).
Fig 3: MAM proteins are reduced in the absence of BOK by biochemical fractionationRepresentative western blot analysis (n = at least 3) for mitochondrial, bulk ER, and MAM proteins in WT and Bok-/- MEFs. Equal total protein was loaded for each fraction (20 µg for IP3R1, 20 µg for IP3R3, 10 µg for CRT, 10 µg for CNX, 40 µg for ANT, 40 µg for VDAC1, and 30 µg for BOK). Molecular weight markers are shown in kDa on the left. Density of the bands in the MAMs was quantified with a Bio-Rad ChemiDoc and plotted on the right. Data are represented as mean ± SD. Significance was calculated using an ANOVA test (ns, non-significant). CNX, calnexin; CRT, calreticulin; Mito, mitochondria; WCL, whole-cell lysate.See also Figure S4.
Fig 4: EBV-LMP1 interacting with ANT1 localizes to mitochondria AKnockdown effect of mPTP complex components.BEffect of knockdown of each mPTP component on the membrane potential of EBV-LMP1-negative or EBV-LMP1-positive nasopharyngeal carcinoma cells. Data are presented as means ± SEM (paired t-test, n = 5, biological replicates per group, *P < 0.05, **P < 0.01).C, DThe specificity of ANT1 antibody was verified in this experiment using: knockdown or overexpression of ANT1 in HK1 cells, respectively (C); expression of HK1-derived ANT1 in prokaryotes and validation after purification (D).ELMP1 is present in the cytoplasm and mitochondria. EBV-LMP1-positive nasopharyngeal carcinoma cells were isolated from cytoplasm and mitochondria and western blot assayed for LMP1 expression.FCo-IP detection of the interaction of EBV-LMP1 and ANT1 in CNE1-LMP1 cells.GLaser confocal analysis of CNE1-LMP1 cells revealed the presence of co-localization of LMP1 with ANT1 (scale bar, 5 µm). The quantified graph on the lower right shows the percentage of yellow fluorescence (merge) to red fluorescence (ANT1) in CNE1-LMP1 cells. Data are presented as means ± SEM (n = 6, biological replicates per group).HPLA detects the presence of direct binding of LMP1 to ANT1, but not VDAC1, in CNE1-LMP1 cells (scale bar, 10 µm).
Fig 5: Over-expression of RCAN1.1S stabilized adenine nucleotide translocator (ANT1) mRNA (a). SH-SY5Y and YD2 cells were treated with 1 µg/mL actinomycin D (Act D) for 0, 3, 6, 9, and 12 h. RT-PCR was used to detect the ANT1 mRNA and dynamin 1 (DNM1) mRNA. 18S rRNA was amplified as an internal control. (b). Quantification of ANT1 mRNA in (a). (c). Quantification of DNM1 mRNA in (a). Values represent mean ± SE; n = 5. mRNA and protein levels at 0 h were artificially set to 100%. (d). ANT1 expression plasmid p3 × flagCMV10-ANT1 was transfected in SH-SY5Y and YD2 cells. A total quantity of 100 µg/mL cycloheximide (CHX) was added 48 h after transfection and cells were collected at 0, 12, 24, 36 h after treatment. Western blot was used to detect the ANT1 protein level using M2 mAb and ß-actin was used as loading control. (e). Quantification of (d). Values represent mean ± SE; n = 5. mRNA and protein levels at 0 h were artificially set to 100%.
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