A-21351

antibody from Invitrogen Antibodies

Targeting: ATP5F1B ATP5B, ATPSB

Provider product page for A-21351

Western blot

Immunocytochemistry

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Antibody data

Antibody Data
Antigen structure
References [83]
Comments [0]
Validations
Western blot [2]
Immunocytochemistry [1]
Flow cytometry [1]
Other assay [48]

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Product number: A-21351 - Provider product page
Provider: Invitrogen Antibodies
Product name: ATP Synthase beta Monoclonal Antibody (3D5AB1)
Antibody type: Monoclonal
Antigen: Other
Description: The antibody was produced in vitro using hybridomas grown in serum-free medium, and then purified by biochemical fractionation. Near homogeneity was judged by SDS-PAGE.
Antibody clone number: 3D5AB1
Concentration: 1 mg/mL

ATP5F1B protein structure - A-21351 shown in red.

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Experimental details: Knockdown of ATP Synthase beta was achieved by transfecting HepG2 cells with ATP Synthase beta specific siRNAs (Silencer® select Product # s1773). Western blot analysis (Fig a) was performed using whole cell extracts from the ATP Synthase beta knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Anti-ATP Synthase beta Monoclonal Antibody (3D5AB1) (Product # A-21351, 1 µg/mL) and Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to ATP Synthase beta.

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Experimental details: Western blot analysis was performed on whole cell extracts of Hep G2 (Lane 1), SK-OV-3 (Lane 2) and, MDA-MB-231 (Lane 3). The blot was probed with Anti- ATP Synthase Subunit beta Mouse Monoclonal Antibody (Product # A-21351, 0.5 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.4 µg/mL, 1:2500 dilution). A ~ 57 kDa band corresponding to ATP Synthase Subunit beta was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody using iBind™ Flex Western Starter Kit (Product # SLF2000S). Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

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Experimental details: Figure 2. Submitochondrial localization of endogenousBcl-x L . (A) Immunoblots of total cell lysates(clarified), cytosolic fractions, and heavy membranes prepared fromdissected cortexes pooled from three 3-d-old mice per sample. Blots wereprobed with antibodies to Bcl-x L (1:1,000; provided by L.Boise), cytochrome c (Cyt c ; 7H8.2C12[1:1,000]), cytochrome oxidase subunit IV (COX IV; 1:1,000; Invitrogen),SMAC (1:1,000; Invitrogen), voltage-dependent anion channel (VDAC;1:1,000; EMD), actin (1:1,000; MP Biomedicals), and UCP-2 (6525 [1:100;Santa Cruz Biotechnology, Inc.]). A representative of three independentexperiments is shown. (B) Summary of immunogold EM staining forBcl-x L and ATP synthase beta proteins in control andcKO mouse brain representing three independent experiments. (C) EM ofmicrodissected CA1 hippocampus (where CA1 synapses onto CA3) from mousebrains stained with gold-labeled Bcl-x L antibody detectsBcl-x L on mitochondrial inner membranes/cristae (blackarrows), outer membrane polar clusters (arrowheads), and adjacentmembranes (line arrows). Bars, 0.1 um. (D) Immunogold staining of bcl-x cKO CA1 mouse brain prepared in parallel asin C. Bar, 0.1 um. (E) Immunoblot analysis of Percoll-purifiednonsynaptic adult rat brain mitochondria. Mitochondria were incubatedwith the indicated proteases (for 30 min) with or without 0.01%digitonin to permeabilize the outer membrane and were blotted forBcl-x L (1:1,000; Abcam), Tom20 (11415 [1:2,000; SantaCruz Biotechnology, Inc.

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Experimental details: Figure 7 Impaired complex V dimerization and complex I supercomplex formation. A) BN-western analysis with beta and alpha subunit antibodies reveals a marked reduction in complex V dimer (VD) relative to monomer (VM) in ATP6 1 mitochondrial isolates compared to wild type controls. B) BN-western analysis using an antibody to complex I (NDUFS3) reveals a loss in normal supercomplexes in ATP6 1 mitochondria. C) Quantitation of the westerns shown in panels A and B comparing WT (green) to ATP6 1 (red): dimer to monomer ratio of complex V and supercomplex to monomer ratio of complex I.

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Experimental details: Figure 2 PINK1 accumulates in the mitochondrial and postmitochondrial supernatant fractions upon knockdown of Lon. (A) Mitochondrial (Mito) and postmitochondrial supernatant (Sup) protein fractions isolated from the heads of flies of the indicated genotypes were analyzed by western blot with antibodies to Myc, Complex V beta (Comp V beta), NADH dehydrogenase (ubiquinone) Fe-S protein 3 (NDUFS3), voltage-dependent anion channel (VDAC) and Actin. elav-GAL4 was used to drive expression of the RNAi constructs used in these experiments. (B) Densitometry of the MPP-PINK1 bands in the mitochondrial fractions from the indicated genotypes was performed using Fiji software. PINK1 band intensities were normalized to VDAC intensity, and this ratio was in turn normalized to the MPP-PINK1/VDAC ratio in Control-R animals. (C) Quantification of the Rho-PINK1 band intensities in mitochondrial fractions from the indicated genotypes was performed as described in B. (D) Quantification of the AFG-PINK1 band intensities in mitochondrial fractions from the indicated genotypes was performed as described in B. (E) Quantification of the MPP-PINK1 band intensities in the supernatant fractions from the indicated genotypes was performed as described in B except that Actin rather than VDAC was used as the loading control. (F) Quantification of the Rho-PINK1 band intensities in supernatant fractions from the indicated genotypes was performed as described in E. (G) Quantification of the AFG-PINK1 band inten

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Experimental details: Figure 3 PINK1 accumulates on the outer membrane and in the matrix upon knockdown of Lon. (A) Mitochondrial fractions isolated from the heads of flies expressing Control-R were divided in half. One of the two samples was treated with 0.5 ug/ml Proteinase K and both samples were incubated at 4degC for 20 minutes. Samples were then subjected to western blot analysis using antibodies to the outer membrane protein Mitofusin (Mfn), the inner membrane-associated matrix protein Complex V beta (Comp V beta), and the matrix luminal protein pyruvate dehydrogenase (PDH). elav-GAL4 was used to drive expression of all the RNAi constructs used in this and the following experiments. (B) Densitometry of Mfn, Comp V beta, and PDH bands from experiments represented by panel A were performed using Fiji software, and the ratios of the band intensities in the sample containing ProK to the sample lacking ProK are shown. (C) Mitochondrial fractions isolated from the heads of flies expressing Control-R and PINK1-Myc were divided in half. Each sample was treated with the indicated concentrations of ProK. Following incubation, samples were subjected to western blot analysis using antibodies to the indicated proteins. (D) Densitometry of the PINK1-Myc, Comp V beta, and PDH bands from experiments represented by panel C were performed as described in B. The Mfn quantification data from panel B is also included for comparison. (E) Mitochondrial fractions isolated from the heads of Lon-R2 and PINK1-Myc exp

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Experimental details: Figure 2 Effects of PAHs on IF1 expression both in vivo and in vitro . ( A ) The levels of IF1 protein and of the beta-F1 subunit of the F0F1ATPase were analyzed by western blotting in liver tissues from rats exposed to 0.04 or 0.8 mg.kg -1 of a 16 PAH mixture. HSC70 was used as loading control. ( B ) IF1 mRNA expression was monitored in F258 cells treated with B[a]P (50 nM or 1 uM) for 24 or 48 hours. ( C ) The IF1 protein content of mitochondrial fractions of B[a]P-exposed F258 cells was evaluated by western blotting. Histogram gives the results of densitometric analysis of IF1 protein level relative to beta-F1 subunit protein level. COXIV was used as loading control. Number of independent experiments = 3 for all conditions. *p < 0.05, **p < 0.01, ***p < 0.001, DMSO vs B[a]P-treated cells, unless otherwise quoted.

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Experimental details: Figure 3 B[a]P metabolism was not involved in IF1 up-regulation in response to B[a]P. F258 cells were pre-treated for 1 hour with alpha-NF (10 uM) prior to co-exposure to B[a]P for 48 hours. The involvement of B[a]P metabolism in IF1 up-regulation was assessed by analyzing IF1 protein level on mitochondrial fractions by western blotting. Histogram gives the results of densitometric analysis of IF1 protein level relative to beta-F1 subunit protein level. COXIV was used as loading control. Results were representative of 3 independent experiments. *p < 0.05, ***p < 0.001, DMSO vs B[a]P-treated cells, unless otherwise quoted.

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Experimental details: Figure 8 Higher level of ATP synthase protein in presynaptic terminals of Pink1/Parkin DKO mice. (A) Representative confocal micrographs of immunohistochemistry for mitochondrial protein ATP synthase beta subunit (ATP Syn) in presynaptic terminals (arrowheads) of tibialis anterior NMJs in the Pink1/Parkin DKO mice at ages between P85 and P110 compared to wild-type mice of the same age. Presynaptic terminals of NMJs were defined by anti-neurofilament and anti-synaptophysin staining pattern (NF + Synp). Postsynaptic acetylcholine receptors were labeled with Alexa 594-conjugated alpha-bungarotoxin (BTX). In these cross-section images of NMJs, presynaptic terminals are above the bungarotoxin signal, and postsynaptic myotubes are below the bungarotoxin signal. Scale bar: 10 mum. (B) Average signal intensity of ATP synthase beta subunit is greater in presynaptic terminals of Pink1/Parkin DKO mice than in those of wild-type mice. Quantifications are from n = four animals and 97-101 NMJs for each genotype in confocal images. Graph shows mean +- SEM. Asterisks indicate significant differences as analyzed by un-paired t -test (* p < 0.05).

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Experimental details: Figure 2 Perturbations of PINK1 and parkin specifically influence dMfn abundance. Protein extracts from wt males (m), PINK1 B9 hemizygous males, wt males and females (m and f) and park 25 males and females were subjected to western blot analysis with an affinity-purified anti-dMfn antiserum (A), an anti-Opa1 antiserum (C), an affinity-purified anti-Drp1 antiserum (D), an anti-complex V beta antiserum (A, C, D), an anti-VDAC antiserum (A, C, D), and an anti-actin antiserum (A, C, D). Arrow in (A) indicates location of the dMfn band at 94 kDa and the asterisks (*) indicate nonspecific bands. (B) Quantification of dMfn abundance in PINK B9 and park 25 mutants relative to wt controls. Because PINK1 B9 males are sterile and the PINK1 gene resides on the X chromosome, we are only able to generate male PINK1 B9 mutants and thus used wt males as the control population for these mutants. The ratio of dMfn to actin abundance was obtained from three independent blots for each sample analyzed; the ratios for wt controls were set at a value of 1 and mutant ratios were normalized to the wt ratios. *p

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Experimental details: Figure 3 Expression and activities of ATP synthase in IF1-KO mouse ( A ) Expression of alpha- and beta-subunits of ATP synthase in various tissues. Whole protein extracts of different tissues from 3-month-old WT and IF1-KO mice were analysed by SDS/PAGE followed by immunoblotting. ( B ) Content of the monomer and dimer forms of ATP synthases in mitochondria. Left panel. Clear Native PAGE. The solubilized mitochondrial protein (5 mug) of the hearts from 8-month-old WT and IF1-KO mice were subjected to Clear Native PAGE and immunoblotted with anti-beta subunit of ATP synthase antibody. Another gel was stained with CBB (Coomassie Brilliant Blue) to show that equal amount of proteins were loaded to PAGE. The positions of molecular mass markers are indicated on the left. Right panel. WT ( n =6) and IF1-KO ( n =5) mice were subjected to the analysis and band intensities were quantified. Dimer content (%) is expressed as 100xdimer/(monomer+dimer). ( C ) Mitochondrial ATP synthesis and hydrolysis activities. Mitochondria were prepared from livers of 4-month-old WT ( n =6) and IF1-KO ( n =5) mice.

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Experimental details: Fig. 3. ND2 mutants exhibit decreased mitochondrial function. (A) Mitochondrial complex-I-dependent respiration rates were measured in mitochondria isolated from middle-aged (14-day-old) or old (30- to 35-day-old) ND2 mutant or controls, revealing age-related declines in both state 3 and maximal state 3 values within genotypes ( P =0.08 and P =0.03 for comparison of middle-aged and old control or ND2 mutant state 3 values, respectively; P =0.001 and P =0.003 for comparison of middle-aged and old control or ND2 mutant maximal state 3 values, respectively). State 4 respiration was similar under all conditions tested. No differences were found for state 3 values between control and ND2 mutants at either middle or old ages. However, maximal state 3 values were decreased in both middle-aged and old ND2 mutants when compared with age-matched controls ( P =0.04 for middle-aged and P =0.003 for old animals). Error bars represent standard deviations; n =3 independent mitochondrial preparations; ** P

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Experimental details: Figure 3. Bcl-x L association with inner mitochondrial membranecomponents. (A) Summary of yeast two-hybrid interactionsbetween BCL-2 family proteins and the ATP synthase beta subunit,including wild-type (WT) and Bcl-x L mutants mt1(F131V/D133A), mt7 (V135A/N136I/W137L), and mt8 (G138E/R139L/I140N). (B)Yeast (BY4741) transformed with the indicated plasmids were heat ramptreated and presented as colony counts from four determinations in twoindependent experiments. Data are presented as the mean +- SEM.Student's t test was used; *, P =0.012 compared with control; **, P = 0.001. (C)Immunoblots for Bcl-x L (provided by L. Boise) or betasubunit in total rat liver mitochondria (T), inner membrane vesicles(F1), and highly purified ATP synthasomes (F2 and F3) of increasingpurity ( Ko et al., 2003 ). Thereplicate blot in the middle was probed with Bcl-x L antibodypreincubated with recombinant Bcl-x L (rBcl-x L )protein purified from Escherichia coli (lanesF1-F3 only). (D) Coomassie blue-stained preparative SDSgel of final purification step for Bcl-x L -bindingpartners from WEHI 7.1 cells. (E) Immunoblots for Bcl-x L andbeta subunit in size column chromatography fractions (F) and totallysates (T). Eluted size markers are indicated.

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Experimental details: Figure 4. A subpopulation of Mff localizes to ER. (A) Endogenous Mff localization in a fixed U2OS cell by immunofluorescence. Cells labeled with anti-Tom20 (mitochondria, blue), anti-PMP70 (peroxisomes, gray), anti-Mff (green) and transfected with ER-TagRFP (ER, red). Left, scrambled siRNA; right, Mff siRNA. Yellow arrows, independent punctae; blue arrow, peroxisome-associated puncta; white arrow, mitochondrially associated Mff. (B) Graph depicting the percentage of colocalization between independent Mff punctae and ER in U2OS cells (endogenous Mff). 54 independent Mff punctae were counted from five ROIs from four cells. Mean values from ROIs: 89.3 +- 6.7%, colocalized Mff with ER; 6.0 +- 6.1%, not colocalized; 4.8 +- 7.3%, unclear localization. (C) Live-cell time-lapse of GFP-Mff-S (green) in U2OS cell also expressing mCherry-mito3 (gray), eBFP2-peroxisome (blue), and E2-Crimson-ER (red). Yellow arrows, independent Mff punctae associating with ER; blue and gray arrows, peroxisomal and mitochondrial Mff, respectively. See also Video 6. (D) Graph depicting the degree of association between independent GFP-Mff-S punctae and ER from live-cell videos as in C (2.5-min videos imaged every 1.5 s). 34 ROIs from 30 U2OS cells analyzed (441 independent Mff punctae). Mean values from ROIs: 86.1 +- 17.1%, stably associated Mff punctae with ER; 11.0 +- 16.8%, partially associated; 4.6 +- 9.4%, not associated. (E) U2OS fractionation. Left: LSP, MSP, and HSP are low, medium, and

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Experimental details: Figure 4 Mitochondrial oxidative phosphorylation and morphology throughout CO generation. ( A ) Bar graph showing the oxidative phosphorylation (OXPHOS) complexes I-V assembly levels in H9 hESCs, H9 hESC COs, PBMCs, iPSCs and iPSC COs. Median fluorescence intensities for each complex were recorded using Luminex technology. Bars, median fluorescence intensity +- SD. ( B ) Representative immunofluorescence images showing the formation of the inner mitochondrial membrane OXPHOS proteins (complex I, NDUFS3; complex II, SDHA; complex III, UQCRC1; complex IV, COXIV; complex V, ATP synthase-beta; green) and the outer mitochondrial membrane (TOMM-20; red) in (i) H9 hESCs to H9 hESC COs and; (ii) iPSCs to iPSC COs. All scale bars, 100 mum. Last column shows insets, enlarged views of boxed areas from the merge CO images, all scale bars, 10 mum. In the CO tissues, out of focus light and autofluorescence of tissue matrix led to background noise which were corrected for better visualization. Brightness levels of each images were also adjusted to optimize visualization. Immunofluorescent images shown here were used for qualitative observations only--no quantitative analyses were performed. ( C ) Bar graph showing the intracellular ATP levels across H9 hESCs to H9 hESC COs and PBMCs, iPSCs to iPSC COs, bars, mean +- SD. ( D ) (i) Box plot summarizing the median number of mitochondria in 10 cells examined, boxes, median and interquartile range, IQR. (ii) Representative electron micrographs o

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Experimental details: Figure 5 Confocal microscopy analysis of autophagosome marker LC3B and mitochondrial marker ATP synthase beta in WT and dKO RPE cells ( a , e ). The colocalization of LC3B ( b , f ) and ATP synthase beta ( c , g ) will show the amount of local autophagosome formation. Our image analysis showed a ~158% increase in the LC3B and ATP synthase beta colocalization in dKO compared to in WT ( d , h , i ). Scale = 5 um. **p < 0.0025.

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Experimental details: Figure 6 Confocal microscopy analysis of lysosomal marker LAMP2 and mitochondrial marker ATP synthase beta in WT and dKO RPE cells ( a , e ). The colocalization of LAMP2 ( b , f ) and ATP synthase beta ( c , g ) shows the amount of autolysosome formation. Image analysis showed no significant changes in LAMP2 and ATP synthase beta colocalization in dKO animals compared to WT ( d , h , i ). Scale = 5 um. p = 0.70. ns-Nonsignificant.

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Experimental details: Figure A1 Representative graphical data from double-staining of AMBRA1 ( a , p = 0.4), Bnip3 ( b , p = 0.4) and FUNDC1 ( c , p = 0.7) with ATP synthase beta. There was no statistically significant difference in the colocalization analysis between one-year-old WT and dKO. ns-Nonsignificant.

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Experimental details: Fig. 6 Autofluorescence and mitochondrial colocalization on the flat-mounted retina from pig and nonhuman primate ( Macaca fascicularis ). Autofluorescence ( a , d ) and mitochondrial ATP-synthase immunolabelling ( b , e ) with the merged images ( c , f ) in cone inner segments ( g ), ganglion cell layer ( h ), and optic fiber layer ( i ) showing co-localization except for the unspecific red labeling of blood vessels in the pig ganglion layer ( h ). Scale bars represent 10 um.

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Experimental details: Figure 4. The biogenesis of several AIs containing nuclear-encoded subunits is stalled when dAIF is disrupted. (A) A representation of mammalian CI showing the approximate relative positions of the 45 subunits. The first four letters of the nuclear-encoded subunit names have been eliminated for simplicity. The N, Q, P P , and P D modules are color-coded in red, orange, blue, and green, respectively. The antibodies used to track the various modules are shown and color-coded accordingly. (B) A schematic depicting the step-wise assembly process of CI. AIs corresponding to the Q, P D , Q+P P , and Q+P P +P D (i.e., Q+P) modules can be tracked by immunoblotting. (C-G) Mitochondrial preparations from flight muscles isolated from adult flies with the genotypes shown were analyzed by BN-PAGE, followed by immunoblotting with the indicated antibodies. (H and I) Total cell lysates from flight muscles isolated from adult flies with the genotypes shown were analyzed by SDS-PAGE and immunoblotting with the CI antibodies indicated (H) and the CIII, CIV, and CV antibodies indicated (I).

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Experimental details: Figure 1 MQC markers increase and mitochondrial content decreases in spinal cord of symptomatic SOD 1-G93A mice A, B Representative Western blot (A) and quantification (B) of p62 associated with mitochondria from spinal cords. Protein levels are normalized by subunit ATPase beta of mitochondria (Complex V). Mitochondrial p62 levels are increased at 120 days (symptomatic stage) in SOD1-G93A spinal cord relative to Non Tg and wild-type SOD1 (wtSOD1). Results are expressed as mean +- SEM relative to Non Tg at 30 days; n = 8 mice (four males and four females). At 120 days, ** P = 0.0018 (Non Tg vs. SOD1-G93A) and ** P = 0.0011 (SOD1-G93A vs. wtSOD1) by paired one-way ANOVA with Tukey's correction. No other statistically significant differences were found (paired Friedman's test with Dunn's correction at 30 days and paired one-way ANOVA with Tukey's correction at 60 and 90 days). C, D Representative Western blot (C) and quantification (D) of Tim23 in homogenates from spinal cords using beta-actin as normalizer. Results are expressed as mean +- SEM and relative to Non Tg at 30 days; n = 6 mice (three males and three females). *** P = 0.0002 (Non Tg vs. SOD1-G93A at 120 days) by paired one-way ANOVA with Tukey's correction. No other statistically significant differences were found (paired Friedman's test with Dunn's correction at 30 days and paired one-way ANOVA with Tukey's correction at 60 and 90 days). Tim23 is decreased in SOD1-G93A spinal cord relative to Non Tg

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Experimental details: Fig. 6. Proteasome inhibition decreases mitochondrial respiration and impairs mitochondrial respiratory complex protein homeostasis in NRK cells. A : digitonin-permeabilized NRK cells (5 x 10 6 ) were treated with vehicle control (DMSO) or the ChT-L proteasome inhibitor bortezomib (BTZ; 10 nM) for 24 h. High-resolution respirometry was used to determine oxygen flux through complexes I, II, and III. Values are expressed as % of control of 4 independent experiments. Differences between groups were compared with an unpaired Student's t- test. *Means are significantly different ( P < 0.05) compared with control. B : ATPase activity of ATP synthase (complex V) was assayed in NRK cells treated with DMSO (vehicle control) or BTZ for 24 h. Renal cell extracts (10 mug) with or without oligomycin (20 muM) were incubated with the enzyme mix in the presence of ATP and NADPH (MitoCheck Complex V Activity Assay kit, Cayman). NADH oxidation rate was monitored at 340 nm for up to 30 min with a BioTek Gen5 microplate reader. Bar graph shows oligomycin-inhibitable ATPase activity expressed as % of control ( n = 3). Differences between groups were compared with a Student's t -test. *Means are significantly different ( P < 0.001). C and D : Western blots of SDHA and ATP5B in soluble ( C ) and insoluble fractions ( D ) of NRK cell extracts after treatment with vehicle control (Con; DMSO) or BTZ (10 nM) for 24 h. Representative blots of 4 independent experiments are shown

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Experimental details: Fig. 1 Comparison of human MAT of the femur (fMAT) with sWAT of the thighs (tsWAT). (a) and (b) Adipocyte size is smaller in fMAT than in tsWAT. (a) Representative merged IF images of whole mount stainings showing adipocytes labelled with perilipin-1 (blue). Nuclei were stained with To-Pro3 (red). Green fluorescence shows active mitochondria stained with ATP synthase beta (ComplexV). Scale bars indicate the size of fMAT (left) and tsWAT (right) adipocytes. (b) Frequency of fMAT (gray) and tsWAT (red) adipocyte size of different donors using indicated scale bars of IF images. Adipocyte median size, number of areas and sample size (n) are indicated in the graph. Samples of four donors pooled from at least three independent experiments. (c) Human fMAT increases with age. Quantitative calculated adipose tissue score [arbitrary units 1-3] of fMAT is plotted against donor age. Each dot point represents one individual. Spearman coefficient (r fMAT ), p value and sample size (n) are indicated in the graph. (d) Representative H&E stains of fMAT (left panel) and tsWAT (right panel) are shown. Red arrows indicate infiltrating cells within the tissues. Scale bars indicate 200 mum for fMAT and 100 mum for tsWAT. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Fig. 1

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Experimental details: Fig. 1 Comparison of protein expression in the brain tissues of female and male mice. Quantitative analysis of western blot (WB) images obtained from 1, 5, and 10-month-old female (F) and male (M) mice showing the relative amounts of Complex I ( a ), Complex II ( b ), ATP synthase ( c ), VDAC1 ( d ), UCP4 ( e ), and beta-actin ( f ) as compared to a brain tissue standard (Std). Representative WB images are shown below the plots. 20 mug of total protein were loaded in each lane. Values represent the means +- SD of data obtained from six animals per group; * p < 0.05, ** p < 0.01, and *** p < 0.001 (mark age differences); # p < 0.05, ## p < 0.01, and ### p < 0.001 (mark sex differences)

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Experimental details: Fig. 2 Comparison of protein expression in brown adipose tissue (BAT) from female and male mice. A quantitative analysis of western blot (WB) images obtained from 1, 5, and 10-month old female (F) and male (M) mice showing the relative amounts of Complex I ( a ), Complex II ( b ), ATP synthase ( c ), VDAC1 ( d ), UCP1 ( e ), UCP3 ( f ), and alpha-actin ( g ) as compared to a BAT tissue standard (Std). Representative WB images are shown below the plots. 20 mug of total protein were loaded in each lane. Values represent the means +- SD of data obtained from six animals per group; * p < 0.05, ** p < 0.01, and *** p < 0.001 (mark age differences); # p < 0.05, ## p < 0.01, and ### p < 0.001 (mark sex differences)

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Experimental details: Fig. 3 Comparison of protein expression in skeletal muscle (SkM) from female and male mice. A quantitative analysis of western blot (WB) images obtained from 1, 5, and 10-month old female (F) and male (M) mice showing the relative amounts of Complex I ( a ), Complex II ( b ), ATP synthase ( c ), VDAC1 ( d ), UCP3 ( e ), and alpha-actin ( f ) as compared to a SkM tissue standard (Std). Representative WB images are shown below the plots. 50 mug of total protein was loaded in each lane. Values represent the means +- SD of data obtained from six animals per group; * p < 0.05, ** p < 0.01, and *** p < 0.001 (mark age differences); # p < 0.05, ## p < 0.01, and ### p < 0.001 (mark sex differences)

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Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 4 Comparison of protein expression in spleen tissue from female and male mice. A quantitative analysis of western blot (WB) images obtained from 1, 5, and 10-month old female (F) and male (M) mice showing the relative amounts of Complex I ( a ), Complex II ( b ), ATP synthase ( c ), VDAC1 ( d ), UCP2 ( e ), and beta-actin ( f ) as compared to a spleen tissue standard (Std). Representative WB images are shown below the plots. 50 mug of total protein was loaded in each lane. Values represent the means +- SD of data obtained from six animals per group; * p < 0.05, ** p < 0.01, and *** p < 0.001 (mark age differences); # p < 0.05, ## p < 0.01, and ### p < 0.001 (mark sex differences)

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 2 Immunofluorescence of primary neurons, astrocytes, microglia, and OPCs derived from WT rats. Cultured cells were treated with 100 nM valinomycin or DMSO control for 4 h at 37 degC, then fixed and analyzed by immunofluorescence with the indicated antibodies. Cells were also stained with DAPI to show cell nuclei. White boxes indicate areas shown at higher magnification immediately below. Each channel is shown individually as well as an image of the DAPI, pS65-Ub, and mitochondrial marker channels merged, as indicated. a Primary cortical neurons treated with DMSO control (left) or with valinomycin (right). Higher-magnification images of the areas indicated by yellow boxes are shown at bottom to clarify the non-overlapping pS65-Ub immunofluorescence with the neuronal marker MAP2A. b Primary cortical astrocytes treated with DMSO control or with valinomycin. A second panel of higher-magnification images of cortical astrocytes treated with valinomycin is included to allow better visualization of the colocalization of pS65-Ub with the mitochondrial marker TIM23 in astrocytes. c Primary cortical microglia treated with DMSO control or with valinomycin. The mitochondrial marker ATP-Synthase was substituted for TIM23 because the microglial marker OX-42 antibody is the same species and isotype as anti-TIM23. d Primary cortical OPCs treated with DMSO control or with valinomycin. Additional higher-magnification images of cortical OPCs treated with valinomycin are shown below to bette