Journal: Nature metabolism

Article Title: Glucose-dependent partitioning of arginine to the urea cycle protects β-cells from inflammation

doi: 10.1038/s42255-020-0199-4

Figure Lengend Snippet: a, Histogram of relative abundance of 3160 proteins detected (out of 5399 total, see ) by LC–MS/MS in 8.7 × 10 4 purified human ²-cells from n = 3 donors. Green lines indicate gene products related to the urea cycle, argininosuccinate and aspartate metabolism detected at the protein level. b, Expression levels of genes related to the urea cycle, pyruvate metabolism and arigninosuccinate shunt based on RNAseq analysis of FACS-sorted human β-cells from n=3 human donors, data are in Log2CPM. c, Co-immunostaining of insulin and individual metabolic enzymes related to the urea cycle in dispersed human islet cells from one donor representing similar results obtained from two additional donors. Representative images are shown (left), scale bar is 10 microns. Mean fluorescence intensity (FI) within the insulin positive region of interest (ROI) was calculated from 5 images per antibody (right). Neg denotes negative control for background Alexa Fluor 488 signal with insulin co-stain. Statistical analyses are student’s t-tests of each enzyme compared to Neg. Enzyme abbreviations in a–c are ARG2, arginase 2; ASL, argininosuccinate lyase; ASS1, argininosuccinate synthase 1; CPS1, carbamoyl-phosphate synthase 1; DDAH 1, dimethylarginine dimethylaminohydrolase 1; DDAH 2, dimethylarginine dimethylaminohydrolase 2; GOT1, aspartate aminotransferase 1; GOT2, aspartate aminotransferase 2; MDH1, malate dehydrogenase 1; MDH2, malate dehydrogenase 2; NAGS, N-acetyl-glutamate synthase; OAT, ornithine aminotransferase; PC, pyruvate carboxylase; SLC25A12, solute carrier family 25 member 12/calcium-binding mitochondrial carrier protein Aralar 1; SLC25A13, solute carrier family 25 member 13/calcium-binding mitochondrial carrier protein Aralar 2; SLC25A15, solute carrier family 25 member 15/mitochondrial ornithine transporter 1. d–e, Cytokine-induced changes in the partitioning of 15 N 2 -L-arginine to urea ( d ) and citrulline/NO ( e ) synthesis in human islets comparing protective vs non-protective glucose metabolism as modeled by BAD SAHB A SD vs RO0281675 treatment, respectively, n=4 donors. f, Chemical inhibition of arginase via ABH interferes with the protective effect of BAD SAHB A SD in human islets undergoing inflammation, n=5 donors. Data are means ± s.e.m. with one-way (d,e) and two-way (f) ANOVA statistical tests with Tukey adjustment for multiple comparisons.

Article Snippet: For all genetic manipulations, corresponding changes in protein expression were verified by western blot analysis using the following antibodies and dilutions; GOT1 (Sino Biological 14196-T52-50, 1:1000), GOT2 (My Bio Source MBS769801, 1:2000), glucokinase (Santa Cruz, 1:1000), pyruvate carboxylase (PCB, Santa Cruz, 1:1000), ARG2 (Life technologies, 1:1000), V5 (Cell signaling, 1:1000), MYC-tag (Cell Signaling, 1:1000), BAD (Abcam, 1:3000), and actin (Sigma, 1:20000).

Techniques: Liquid Chromatography with Mass Spectroscopy, Purification, Expressing, Immunostaining, Fluorescence, Negative Control, Staining, Binding Assay, Inhibition

Journal: Nature metabolism

Article Title: Glucose-dependent partitioning of arginine to the urea cycle protects β-cells from inflammation

doi: 10.1038/s42255-020-0199-4

Figure Lengend Snippet: a, Schematic of the TCA and urea cycles and their connection via the aspartate-argininosuccinate shunt. Enzymes of interest are marked in red and their corresponding inhibitors in blue. b, Total aspartate levels in human islets treated with the indicated compounds and cultured in the absence or presence of inflammatory cytokines. Data are from the untargeted metabolomics analysis in , n=5 human donors pooled and split into 8 replicates. c, Contribution of glucose to total aspartate pools in mouse islets labeled with 13 C 6 glucose and treated with inflammatory cytokines in the context of protective vs non-protective glucose metabolism. Data are from n=5 (Veh, RO0281675), n=6 (BAD SAHB A SD) and n=4 (BAD SAHB A AAA) independent mouse islet isolations and experiments. See for isotopologue distribution of aspartate in an analogous labelling experiment. d–e, Quantification of urea and NO , and viability ( e ) in human islets subjected to shRNA-mediated GOT1 ( G1 ) or GOT2 ( G2 ) depletion and treated with cytokines in the context of protective vs non-protective glucose metabolism, n=4 donors for urea, n=3 donors for NO, and n=5 donors for viability measurements. Data for one hairpin per gene are displayed (sh#1 for GOT1 and sh#2 for GOT2 ) from the full data set of multiple hairpins, see – . f–g, Quantification of urea levels ( f ) and viability ( g ) in human islets supplemented with argininosuccinate (AS) in the presence of inflammatory cytokines, H 2 O serves as vehicle control for AS. Data are from 6 independent experiments from n=3 donors. Data are means ± s.e.m. with statistical analyses on means from independent experiments using one-way ANOVA with Tukey adjustment for multiple comparisons.

Article Snippet: For all genetic manipulations, corresponding changes in protein expression were verified by western blot analysis using the following antibodies and dilutions; GOT1 (Sino Biological 14196-T52-50, 1:1000), GOT2 (My Bio Source MBS769801, 1:2000), glucokinase (Santa Cruz, 1:1000), pyruvate carboxylase (PCB, Santa Cruz, 1:1000), ARG2 (Life technologies, 1:1000), V5 (Cell signaling, 1:1000), MYC-tag (Cell Signaling, 1:1000), BAD (Abcam, 1:3000), and actin (Sigma, 1:20000).

Techniques: Cell Culture, Labeling, shRNA

Journal: Nature metabolism

Article Title: Glucose-dependent partitioning of arginine to the urea cycle protects β-cells from inflammation

doi: 10.1038/s42255-020-0199-4

Figure Lengend Snippet: a, 13 C fractional labelling of aspartate from 13 C 6 glucose. Data are shown as non-normalized to vehicle PBS and display the fraction of each M+ n mass isotopomer out of the total pool of aspartate for each condition. For clarity, statistical comparisons are only shown for each M+ n of a given condition (RO0281675, BAD SAHB A SD and BAD SAHB A AAA) compared to the corresponding M+ n of vehicle control. Data are pooled means from n=6 (Veh), n=5 (RO0281675), and n=6 (BAD SAHB A SD, BAD SAHB A AAA) independent mouse islet isolations and experiments. b, Western blot analysis of GOT1/2 knockdown efficiency using multiple independent hairpins for data shown in – and – . Blots are representative of n=2 independent experiments with similar results. c–d, Aspartate ( c ), urea and NO ( d ) levels in human islets from the same experiments shown in – , displaying the complete set of data on all hairpins tested. Aspartate data are from n = 4 human donors for shCtrl samples and n = 3 donors for knockdown samples. Urea and NO data are from n = 4 and n = 3 donors, respectively. Statistical analyses in (a) are two-way ANOVA showing p-value comparisons for each condition to Veh, and one-way ANOVA in (c–d), both with Tukey adjustment for multiple comparisons.

Article Snippet: For all genetic manipulations, corresponding changes in protein expression were verified by western blot analysis using the following antibodies and dilutions; GOT1 (Sino Biological 14196-T52-50, 1:1000), GOT2 (My Bio Source MBS769801, 1:2000), glucokinase (Santa Cruz, 1:1000), pyruvate carboxylase (PCB, Santa Cruz, 1:1000), ARG2 (Life technologies, 1:1000), V5 (Cell signaling, 1:1000), MYC-tag (Cell Signaling, 1:1000), BAD (Abcam, 1:3000), and actin (Sigma, 1:20000).

Techniques: Western Blot

Journal: Molecular Neurobiology

Article Title: Exosome-Derived lncRNA NEAT1 Exacerbates Sepsis-Associated Encephalopathy by Promoting Ferroptosis Through Regulating miR-9-5p/ TFRC and GOT1 Axis

doi: 10.1007/s12035-022-02738-1

Figure Lengend Snippet: miR-9-5p, sponged by NEAT1, overexpressed in vitro and in vivo and might promote cell apoptosis in sepsis-induced ferroptosis by targeting TFRC and GOT1 A The binding regions between miR-9-5p with sequences from NEAT1, TFRC , and GOT1 . B , C The expression level of miR-9-5p, TFRC , and GOT1 in model and control was tested by qRT-PCR in vivo (normalized to U6 or GAPDH ). *** P < 0.001 vs control. D The protein levels of TFRC and GOT1 in cerebral cortex were assessed using Western blotting. E The protein level of TFRC in cerebral cortex was showed by immunohistochemistry. F , G The dual-luciferase reporter gene assay analyzes the binding relationship between miR-9-5p with sequences from NEAT1, TFRC , and GOT1 . *** P < 0.001 vs NC. H , I The expression level of miR-9-5p, TFRC , and GOT1 in iron-rich (100 μM FeCl 3 ), control (100 μM FeCl 3 + control serum), and model (100 μM FeCl 3 + sepsis serum) group was tested by qRT-PCR (normalized to U6 or GAPDH ). * P < 0.05, ** P < 0.01, *** P < 0.001 vs iron-rich and control. J The protein levels of TFRC and GOT1 assessed using western blotting in vitro. K , L The immunofluorescence for detecting TFRC and GOT1 (blue, DAPI; green, TFRC; red, GOT1).

Article Snippet: Subsequently, the samples was incubated with primary antibodies, including rabbit anti-mouse polyclonal antibodies to TSG01 (1:2000; ab125011), CD9 (1:2000; ab92726), CD63 (1:2000; ab193349), TFRC (1:2000; ab214039), GOT1 (1:1000; ab221939) and GAPDH (1:5,000; ab8245) for 1 h. The samples were then incubated in the secondary antibody, horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:20,000, BA1054, BOSTER Inc.) with oscillation at room temperature for 40 min.

Techniques: In Vitro, In Vivo, Binding Assay, Expressing, Control, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Luciferase, Reporter Gene Assay, Immunofluorescence

Journal: Molecular Neurobiology

Article Title: Exosome-Derived lncRNA NEAT1 Exacerbates Sepsis-Associated Encephalopathy by Promoting Ferroptosis Through Regulating miR-9-5p/ TFRC and GOT1 Axis

doi: 10.1007/s12035-022-02738-1

Figure Lengend Snippet: Increased miR-9-5p regulates the expression of TFRC and GOT1 and relieves ferroptosis in vitro The bEnd.3 cells were transfected with miR-9-5p angomir, followed by serum stimulation. A The expression levels of NEAT1, miR-9-5p, TFRC , and GOT1 in control, model, and model + miR-9-5p angomir were tested by qRT-PCR (normalized to U6 or GAPDH ). B The sequence for NEAT1 mutant. C The protein levels of TFRC and GOT1. D , E The immunofluorescence for TFRC and GOT1 (blue, DAPI; green, TFRC; red, GOT1). F The cell viability of bEnd.3 cells were evaluated by CCK-8 assay. G – K The levels of ROS, Fe ion (Fe 2+ and Fe 3+ ), GSH, GPX4, and MDA were assessed. * P < 0.05, ** P < 0.01, *** P < 0.001 vs control; # P < 0.05, ## P < 0.01, ### P < 0.001 vs model.

Article Snippet: Subsequently, the samples was incubated with primary antibodies, including rabbit anti-mouse polyclonal antibodies to TSG01 (1:2000; ab125011), CD9 (1:2000; ab92726), CD63 (1:2000; ab193349), TFRC (1:2000; ab214039), GOT1 (1:1000; ab221939) and GAPDH (1:5,000; ab8245) for 1 h. The samples were then incubated in the secondary antibody, horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:20,000, BA1054, BOSTER Inc.) with oscillation at room temperature for 40 min.

Techniques: Expressing, In Vitro, Transfection, Control, Quantitative RT-PCR, Sequencing, Mutagenesis, Immunofluorescence, CCK-8 Assay

Journal: Molecular Neurobiology

Article Title: Exosome-Derived lncRNA NEAT1 Exacerbates Sepsis-Associated Encephalopathy by Promoting Ferroptosis Through Regulating miR-9-5p/ TFRC and GOT1 Axis

doi: 10.1007/s12035-022-02738-1

Figure Lengend Snippet: Enhanced NEAT1 promoted ferroptosis and increased the expression of TFRC and GOT1 in vitro NEAT1 was overexpressed in bEnd.3 cells. A The expression levels of NEAT1, miR-9-5p, TFRC , and GOT1 in Vector, WT NEAT1 (wild NEAT1 sequence), and MUT NEAT1 (site-directed mutant NEAT1) by qRT-PCR (normalized to U6 or GAPDH ). B The protein levels of TFRC and GOT1 showed by western blotting. C , D The immunofluorescence for TFRC and GOT1 (blue, DAPI; green, TFRC; red, GOT1). E The cell viability of cells detected by CCK-8 assay. F – J The levels of ROS, Fe ion (Fe 2+ and Fe 3+ ), GSH, GPX4, and MDA. ** P < 0.01, ** P < 0.01, *** P < 0.001 vs Vector; ## P < 0.01, ### P < 0.001 vs WT NEAT1.

Article Snippet: Subsequently, the samples was incubated with primary antibodies, including rabbit anti-mouse polyclonal antibodies to TSG01 (1:2000; ab125011), CD9 (1:2000; ab92726), CD63 (1:2000; ab193349), TFRC (1:2000; ab214039), GOT1 (1:1000; ab221939) and GAPDH (1:5,000; ab8245) for 1 h. The samples were then incubated in the secondary antibody, horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:20,000, BA1054, BOSTER Inc.) with oscillation at room temperature for 40 min.

Techniques: Expressing, In Vitro, Plasmid Preparation, Sequencing, Mutagenesis, Quantitative RT-PCR, Western Blot, Immunofluorescence, CCK-8 Assay

Journal: Molecular Neurobiology

Article Title: Exosome-Derived lncRNA NEAT1 Exacerbates Sepsis-Associated Encephalopathy by Promoting Ferroptosis Through Regulating miR-9-5p/ TFRC and GOT1 Axis

doi: 10.1007/s12035-022-02738-1

Figure Lengend Snippet: miR-9-5p suppressed the expression of TFRC and GOT1 in vivo A – D The qRT-PCR analysis on the expression of NEAT1, miR-9-5p, TFRC , and GOT1 in the cerebral cortex of control, model, and model + miR-9-5p angomir rats, respectively (normalized to U6 or GAPDH ). E The western blotting analysis on TFRC and GOT1. F The protein level of TFRC in four brain regions were assessed by Immunohistochemistry. *** P < 0.001 vs control; ### P < 0.001 vs model.

Article Snippet: Subsequently, the samples was incubated with primary antibodies, including rabbit anti-mouse polyclonal antibodies to TSG01 (1:2000; ab125011), CD9 (1:2000; ab92726), CD63 (1:2000; ab193349), TFRC (1:2000; ab214039), GOT1 (1:1000; ab221939) and GAPDH (1:5,000; ab8245) for 1 h. The samples were then incubated in the secondary antibody, horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:20,000, BA1054, BOSTER Inc.) with oscillation at room temperature for 40 min.

Techniques: Expressing, In Vivo, Quantitative RT-PCR, Control, Western Blot, Immunohistochemistry

Journal: Molecular Neurobiology

Article Title: Exosome-Derived lncRNA NEAT1 Exacerbates Sepsis-Associated Encephalopathy by Promoting Ferroptosis Through Regulating miR-9-5p/ TFRC and GOT1 Axis

doi: 10.1007/s12035-022-02738-1

Figure Lengend Snippet: miR-9-5p suppressed the expression of and GOT1 in vivo The protein level of GOT1 in four brain regions was assessed by Immunohistochemistry.

Article Snippet: Subsequently, the samples was incubated with primary antibodies, including rabbit anti-mouse polyclonal antibodies to TSG01 (1:2000; ab125011), CD9 (1:2000; ab92726), CD63 (1:2000; ab193349), TFRC (1:2000; ab214039), GOT1 (1:1000; ab221939) and GAPDH (1:5,000; ab8245) for 1 h. The samples were then incubated in the secondary antibody, horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:20,000, BA1054, BOSTER Inc.) with oscillation at room temperature for 40 min.

Techniques: Expressing, In Vivo, Immunohistochemistry

Journal: bioRxiv

Article Title: Cold-inducible GOT1 activates the malate-aspartate shuttle in brown adipose tissue to support fuel preference for fatty acids

doi: 10.1101/2024.11.18.623867

Figure Lengend Snippet: (A) A schematic of the malate-aspartate shuttle (MAS), which consists of two pairs of cytosolic (GOT1 and MDH1) and mitochondrial (GOT2 and MDH2) enzymes and two mitochondrial transporters. MAS transports reducing equivalents produced in glycolysis from the cytosol to mitochondria in the form of malate, which is oxidized back to oxaloacetate (OAA), regenerating NADH in the mitochondria. (B) qPCR analysis to determine the effect of cold stress on MAS gene expression in BAT. BL6 mice were housed at 23°C or exposed to 4°C for 5h. (C) Effect of β 3 -adrenergic stimulation on MAS gene expression in BAT. BL6 mice were administered with vehicle or a β 3 AR agonist CL316243 (1 mg/kg BW) for 5h. (D) Expression of GOT1 protein in brain (B), liver (L), heart (H), muscle (M), kidney (K), brown adipose (BA), and white adipose (WA) tissue. (E) Cold-dependent induction of GOT1 protein in BAT. (F) Increased GOT activity in cold-activated BAT. (G) Effect of cold stress on the MAS enzyme levels in IWAT. BL6 mice were housed at 23°C or exposed to 4°C for 7 days. All data are presented as the Mean ± SEM. ** p <0.01, **** p <0.0001.

Article Snippet: Lysates were separated by SDS-PAGE, transferred to nitrocellulose membrane, and analyzed by immunoblotting with following antibodies: GOT1 antibody (Pro Sci, #30-379), GOT2 antibody (Invitrogen, #PA5-27572), MDH1 antibody (Santa Cruz, #sc-166879), MDH2 antibody (Invitrogen, #PA5-21700), UCP1 antibody , GAPDH antibody (Abcam, #ab9485), VDAC antibody (Abcam, #ab15895), α-tubulin antibody (Abcam, #ab7291), β-actin antibody (Sigma, #A5441), phospho-PDH E1α antibody (S232) (Millipore, #AP106350).

Techniques: Produced, Expressing, Activity Assay

Journal: bioRxiv

Article Title: Cold-inducible GOT1 activates the malate-aspartate shuttle in brown adipose tissue to support fuel preference for fatty acids

doi: 10.1101/2024.11.18.623867

Figure Lengend Snippet: (A, B) Upregulation of Got1 gene expression in brown adipocytes treated with 5 µM isoproterenol (ISO) or 1 mM dibutyryl cAMP for 4h. (C) Enrichment of PGC-1α/NT-PGC-1α at the GOT1 gene promoter in cold-activated BAT. The PGC-1α/NT-PGC-1α ChIP-seq peak was visualized by the Integrative Genome Viewer (IGV) v2.3. (D) PCR analysis of PGC-1α/NT-PGC-1α recruitment to the ERRE of the GOT1 promoter. ChIP was carried out with PGC-1α antibody in nuclear extracts of BAT extracted from mice exposed to 4°C for 5h. (E) Effect of single or combined loss of full-length (FL)-PGC-1α and NT-PGC-1α on Got1 gene expression. Fully differentiated brown adipocytes were treated with 1 mM dibutyryl cAMP for 4h (n=4/group). (F) Effect of ectopic expression of FL-PGC-1α or NT-PGC-1α on Got1 expression in brown adipocytes (n=4/group). (G) Schematic presentation of transcriptional regulation of the Got1 gene by cold or β-AR agonist in brown adipocytes. All data are presented as the Mean ± SEM. # p <0.0001.

Article Snippet: Lysates were separated by SDS-PAGE, transferred to nitrocellulose membrane, and analyzed by immunoblotting with following antibodies: GOT1 antibody (Pro Sci, #30-379), GOT2 antibody (Invitrogen, #PA5-27572), MDH1 antibody (Santa Cruz, #sc-166879), MDH2 antibody (Invitrogen, #PA5-21700), UCP1 antibody , GAPDH antibody (Abcam, #ab9485), VDAC antibody (Abcam, #ab15895), α-tubulin antibody (Abcam, #ab7291), β-actin antibody (Sigma, #A5441), phospho-PDH E1α antibody (S232) (Millipore, #AP106350).

Techniques: Expressing, ChIP-sequencing

Journal: bioRxiv

Article Title: Cold-inducible GOT1 activates the malate-aspartate shuttle in brown adipose tissue to support fuel preference for fatty acids

doi: 10.1101/2024.11.18.623867

Figure Lengend Snippet: (A) Generation of BAT-specific Got1 knock-in mice. The transgene of CAG-loxP-neo-3xpA(stop)-loxP-Got1 was inserted into the Rosa26 locus. Cre-mediated excision of the stop cassette enables targeted expression of Got1 in BAT. (B) qPCR analysis for validation of BAT-specific Got1 expression in R26- Got1 BATOE/+ mice housed at 23°C. (C) Cold-independent elevation of GOT1 protein in BAT of R26- Got1 BATOE/+ male mice at 23°C. (D) No alteration of other MAS enzyme expression by GOT1 overexpression in BAT. (E) H&E staining of BAT from mice at 23°C. Black bars represent 100 µm. (F) Measurement of NAD + /NADH ratios in BAT homogenates. (G-K) Measurement of body weight, energy expenditure, RER, food intake, and locomotor activity in R26- Got1 fl/+ and R26- Got1 BATOE/+ female mice (n=5/group). Mice were placed in indirect calorimetric chambers and monitored prior to and during administration of a β 3 AR agonist CL316243 (1mg/kg BW, daily). (L) Improved cold tolerance of R26- Got1 BATOE/+ female mice (n=5/group). The body temperature was measured with a rectal thermometer. (M) Ucp1 mRNA and protein levels in BAT. (N, O) Glucose tolerance tests of R26- Got1 fl/+ and R26- Got1 BATOE/+ female mice at 23°C (n=7/group) and at 4°C (n=7/group). (P) Measurement of [ 3 H]-2DG uptake by BAT extracted from mice housed at 23°C. (Q) qPCR analysis of BAT extracted from R26- Got1 fl/+ and R26- Got1 BATOE/+ mice housed at 23°C or 4°C (n=6-7/group). All data are presented as the Mean ± SEM. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001, # p <0.0001.

Article Snippet: Lysates were separated by SDS-PAGE, transferred to nitrocellulose membrane, and analyzed by immunoblotting with following antibodies: GOT1 antibody (Pro Sci, #30-379), GOT2 antibody (Invitrogen, #PA5-27572), MDH1 antibody (Santa Cruz, #sc-166879), MDH2 antibody (Invitrogen, #PA5-21700), UCP1 antibody , GAPDH antibody (Abcam, #ab9485), VDAC antibody (Abcam, #ab15895), α-tubulin antibody (Abcam, #ab7291), β-actin antibody (Sigma, #A5441), phospho-PDH E1α antibody (S232) (Millipore, #AP106350).

Techniques: Knock-In, Expressing, Over Expression, Staining, Activity Assay

Journal: bioRxiv

Article Title: Cold-inducible GOT1 activates the malate-aspartate shuttle in brown adipose tissue to support fuel preference for fatty acids

doi: 10.1101/2024.11.18.623867

Figure Lengend Snippet: (A) qPCR analysis for adipogenesis in fully differentiated Got1 fl/fl and Got1 OE brown adipocytes. (B) WB analysis of GOT1 in the whole cell extracts, cytosolic, and mitochondrial fractions. (C) Schematic showing the effect of AOA-dependent inhibition of GOT1 and GOT2 in the MAS. Red arrows indicate the effect of AOA on NADH/NAD + states. (D) Measurement of GOT1 enzyme activity in the cytosolic fraction of brown adipocytes treated with vehicle or 2 mM AOA. (E) Schematic showing Peredox-mCherry, a fluorescent sensor of the NADH-NAD + redox state (left panel). A circularly permuted GFP T-Sapphire (green) is interposed between the two Rex subunits (gray), with an RFP mCherry (red) to normalize for the GFP fluorescence. NADH binding to Rex increases GFP fluorescence. A representative green and red fluorescence profile of Peredox-mCherry (right panel) prior to and after addition of 2 mM AOA, followed by addition of 20 mM pyruvate and 0.4 mM glycolytic inhibitor, iodoacetate. The Peredox signal was normalized to the lowest value with pyruvate and iodoacetate. (F) Effect of GOT1 overexpression on the cytosolic NADH-NAD + redox state measured by the normalized green to red fluorescence ratio of Peredox-mCherry. Approximately 50 cells per group were analyzed. (G) Measurement of mitochondrial uncoupled respiration in brown adipocytes in the presence of an ATPase inhibitor, oligomycin. (H) Schematic of [ 2 H] labeling used to determine cytosolic NADH-oxidizing pathways. The deuterium of NAD 2 H produced from [4- 2 H] glucose can be incorporated into malate, G3P, or lactate. (I) Total and [ 2 H]-labeled NADH levels in Got1 fl/fl and Got1 OE brown adipocytes incubated with 25mM [4- 2 H] glucose at 2.5, 5, and 10 min. (J) Total and [M+1]-malate levels and fraction of [M+1]-malate after introducing [4- 2 H] glucose. (K) Total and [M+1]-, [M+2]-G3P levels and fraction of [M+1]- and [M+2]-G3P after introducing [4- 2 H] glucose. (L) Total and [M+1]-lactate levels and fraction of [M+1]-lactate after introducing [4- 2 H] glucose. All data are presented as the Mean ± SEM. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001, # p <0.0001.

Article Snippet: Lysates were separated by SDS-PAGE, transferred to nitrocellulose membrane, and analyzed by immunoblotting with following antibodies: GOT1 antibody (Pro Sci, #30-379), GOT2 antibody (Invitrogen, #PA5-27572), MDH1 antibody (Santa Cruz, #sc-166879), MDH2 antibody (Invitrogen, #PA5-21700), UCP1 antibody , GAPDH antibody (Abcam, #ab9485), VDAC antibody (Abcam, #ab15895), α-tubulin antibody (Abcam, #ab7291), β-actin antibody (Sigma, #A5441), phospho-PDH E1α antibody (S232) (Millipore, #AP106350).

Techniques: Inhibition, Activity Assay, Fluorescence, Binding Assay, Over Expression, Labeling, Produced, Incubation

Journal: bioRxiv

Article Title: Cold-inducible GOT1 activates the malate-aspartate shuttle in brown adipose tissue to support fuel preference for fatty acids

doi: 10.1101/2024.11.18.623867

Figure Lengend Snippet: (A) Extracellular acidification rates (ECAR) measured by a Seahorse XF analyzer in Got1 fl/fl and Got1 OE brown adipocytes (n=10/group) at baseline and after addition of 10 mM glucose, followed by addition of 10 µM oligomycin and 50 mM 2-DG. (B) Measurement of [ 3 H]-2DG uptake by Got1 fl/fl and Got1 OE brown adipocytes treated with vehicle or 5µM isoproterenol (ISO) for 4h. (C) WB analysis of GLUT1 protein expression. (D) qPCR analysis of genes involved in glucose uptake, glycolysis, and FA oxidation. (E, F) Oxidation of 14 C-labeled pyruvate or palmitate in Got1 fl/fl and Got1 OE brown adipocytes treated with vehicle or isoproterenol for 4h. CO 2 production was normalized by protein concentrations. (G) Mitochondrial respiration in brown adipocytes in the absence and presence of an ATPase inhibitor, oligomycin. (H) The ADP/ATP ratios measured in Got1 fl/fl and Got1 OE brown adipocyte lysates. (I, J) Increased phosphorylation of AMPKα at Thr172 in Got1-overexpressing brown adipocytes and BAT. (K) Schematic showing the effect of GOT1 overexpression on FAO, glucose uptake, and glycolysis. All data are presented as the Mean ± SEM. * p <0.05, ** p <0.01, *** p <0.001, # p <0.0001.

Article Snippet: Lysates were separated by SDS-PAGE, transferred to nitrocellulose membrane, and analyzed by immunoblotting with following antibodies: GOT1 antibody (Pro Sci, #30-379), GOT2 antibody (Invitrogen, #PA5-27572), MDH1 antibody (Santa Cruz, #sc-166879), MDH2 antibody (Invitrogen, #PA5-21700), UCP1 antibody , GAPDH antibody (Abcam, #ab9485), VDAC antibody (Abcam, #ab15895), α-tubulin antibody (Abcam, #ab7291), β-actin antibody (Sigma, #A5441), phospho-PDH E1α antibody (S232) (Millipore, #AP106350).

Techniques: Expressing, Labeling, Over Expression

Journal: bioRxiv

Article Title: Cold-inducible GOT1 activates the malate-aspartate shuttle in brown adipose tissue to support fuel preference for fatty acids

doi: 10.1101/2024.11.18.623867

Figure Lengend Snippet: (A) Generation of BAT-specific Got1 knockout mice. Exon 2 of the mouse GOT1 gene was targeted for deletion by the LoxP/Cre system. (B) qPCR analysis for detection of Got1 transcripts in Got1 fl/fl and Got1 BATKO male mice exposed to 4°C for 7 days (n=6-7/group). (C) WB analysis for GOT1 in Got1 fl/fl and Got1 BATKO mice housed at 23°C or exposed to 4°C for 7 days. (D) Body temperature of Got1 fl/fl and Got1 BATKO female mice during cold exposure (n=7-8/group). Core body temperature was measured using a rectal thermometer at indicated times. (E) WB and qPCR of UCP1 in BAT from the same mice described in D. (F) Glucose tolerance test of cold exposed- Got1 fl/fl and Got1 BATKO female mice (n=7-10/group). (G) Measurement of [ 3 H]-2DG uptake by cold-activated BAT. (H) The NAD + /NADH ratio in BAT homogenates. (I-M) Measurement of body weight, energy expenditure, RER, food intake, and locomotor activity in Got1 fl/fl and Got1 BATKO male mice (n=6/group). Mice were placed in indirect calorimetric chambers and monitored during β-adrenergic stimulation of BAT with a β 3 AR agonist CL316243 (1mg/kg BW, daily). (N) Oxidation of 14 C-labeled palmitate or pyruvate in BAT homogenates. BAT was extracted from mice exposed to 4°C for 7 days. (O) qPCR analysis of genes involved in glucose uptake, glycolysis, and FA oxidation in cold-activated BAT (n=6/group). (P) Uptake of 14 C-labeled pyruvate by the mitochondria isolated from cold-activated BAT. (Q) Reduced phosphorylation of the PDH E1α subunit at Ser232 with a concurrent increase in PDH activity in Got1-deficient BAT. (R) Measurement of pyruvate-dependent mitochondrial respiration in BAT explants extracted from mice exposed to 4°C for 7 days. All data are presented as the Mean ± SEM. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001, # p <0.0001.

Article Snippet: Lysates were separated by SDS-PAGE, transferred to nitrocellulose membrane, and analyzed by immunoblotting with following antibodies: GOT1 antibody (Pro Sci, #30-379), GOT2 antibody (Invitrogen, #PA5-27572), MDH1 antibody (Santa Cruz, #sc-166879), MDH2 antibody (Invitrogen, #PA5-21700), UCP1 antibody , GAPDH antibody (Abcam, #ab9485), VDAC antibody (Abcam, #ab15895), α-tubulin antibody (Abcam, #ab7291), β-actin antibody (Sigma, #A5441), phospho-PDH E1α antibody (S232) (Millipore, #AP106350).

Techniques: Knock-Out, Activity Assay, Labeling, Isolation

Journal: bioRxiv

Article Title: Cold-inducible GOT1 activates the malate-aspartate shuttle in brown adipose tissue to support fuel preference for fatty acids

doi: 10.1101/2024.11.18.623867

Figure Lengend Snippet: (A) Validation of loss of GOT1 expression and its enzymatic activity in the cytosolic fraction in Got1 -deficient brown adipocytes treated with 5µM isoproterenol (ISO) for 4h. (B) qPCR analysis for adipogenic gene expression in Got1 fl/fl and Got1 KO brown adipocytes treated with ISO for 4h. (C) Schematic showing the effect of Got1 deletion on the MAS-mediated NADH shuttling (left penal). Measurement of the cytosolic NADH-NAD + redox state by Peredox-mCherry in Got1 -deficient brown adipocytes (right panel). The green and red fluorescence signals of Peredox-mCherry were measured prior to and after addition of 2 mM AOA, followed by addition of 20 mM pyruvate and 0.4 mM glycolytic inhibitor, iodoacetate. (D) Schematic of [4- 2 H] glucose tracing. (E) [ 2 H]-labeled malate, G3P, and lactate after introducing [4- 2 H] glucose to Got1 fl/fl and Got1 KO brown adipocytes treated with ISO for 4h. (F) [ 3 H]-2DG uptake by brown adipocytes treated with ISO for 4h. (G, H) Oxidation of 14 C-labeled palmitate or pyruvate in brown adipocytes treated with vehicle or ISO for 4h and 48h. (I, J) Mitochondrial respiration in brown adipocytes treated with ISO for 4h or 48h. (K) qPCR analysis of gene expression in brown adipocytes treated with ISO for 48h. (L) WB analysis of Got1 fl/fl and Got1 KO brown adipocytes treated with ISO for 48h. (M) [ 3 H]-2DG uptake by brown adipocytes treated with vehicle or ISO for 48h. (N) Extracellular acidification rates (ECAR) in Got1 fl/fl and Got1 OE brown adipocytes (n=5/group) at baseline and after addition of 10 mM glucose, followed by addition of 10 µM oligomycin and 50 mM 2-DG. (O) A schematic diagram describing the role of GOT1 in cold-activated brown adipocytes. Cold-inducible GOT1 acts as a critical node that links cold-stimulated βAR signaling to the malate-aspartate shuttle (MAS). GOT1-dependent MAS activation facilitates the transport of reducing equivalents produced in glycolysis from the cytosol to mitochondria in the form of malate. This process is essential for the efficient oxidation of fatty acids (FA) to support thermogenesis under cold stress. Got1-deficient brown adipocytes rewire mitochondrial fuel preference toward glucose by increasing mitochondrial pyruvate import and PDH-mediated conversion of pyruvate to acetyl-CoA. All data are presented as the Mean ± SEM. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001, # p <0.0001.

Article Snippet: Lysates were separated by SDS-PAGE, transferred to nitrocellulose membrane, and analyzed by immunoblotting with following antibodies: GOT1 antibody (Pro Sci, #30-379), GOT2 antibody (Invitrogen, #PA5-27572), MDH1 antibody (Santa Cruz, #sc-166879), MDH2 antibody (Invitrogen, #PA5-21700), UCP1 antibody , GAPDH antibody (Abcam, #ab9485), VDAC antibody (Abcam, #ab15895), α-tubulin antibody (Abcam, #ab7291), β-actin antibody (Sigma, #A5441), phospho-PDH E1α antibody (S232) (Millipore, #AP106350).

Techniques: Expressing, Activity Assay, Fluorescence, Labeling, Activation Assay, Produced

Journal: bioRxiv

Article Title: Cold-inducible GOT1 activates the malate-aspartate shuttle in brown adipose tissue to support fuel preference for fatty acids

doi: 10.1101/2024.11.18.623867

Figure Lengend Snippet: (A) Total and labeled M+1 malate levels and fraction of labeled malate after introducing [4- 2 H] glucose. (B) Total and labeled G3P levels and fraction of labeled M+1 G3P and M+2 G3P after introducing [4- 2 H] glucose. (C) Total and labeled M+1 lactate levels and fraction of labeled lactate after introducing [4- 2 H] glucose. Got1 fl/fl and Got1 KO brown adipocytes were treated with isoproterenol for 4h prior to addition of 25 mM [4- 2 H] glucose. The middle panels showing the labeled metabolites are also presented in Figure 7E. All data are presented as the Mean ± SEM. * p <0.05, ** p <0.01.

Article Snippet: Lysates were separated by SDS-PAGE, transferred to nitrocellulose membrane, and analyzed by immunoblotting with following antibodies: GOT1 antibody (Pro Sci, #30-379), GOT2 antibody (Invitrogen, #PA5-27572), MDH1 antibody (Santa Cruz, #sc-166879), MDH2 antibody (Invitrogen, #PA5-21700), UCP1 antibody , GAPDH antibody (Abcam, #ab9485), VDAC antibody (Abcam, #ab15895), α-tubulin antibody (Abcam, #ab7291), β-actin antibody (Sigma, #A5441), phospho-PDH E1α antibody (S232) (Millipore, #AP106350).

Techniques: Labeling

Journal: bioRxiv

Article Title: Cold-inducible GOT1 activates the malate-aspartate shuttle in brown adipose tissue to support fuel preference for fatty acids

doi: 10.1101/2024.11.18.623867

Figure Lengend Snippet: (A) H&E staining analysis of BAT and IWAT from Got1 fl/fl and Got1 BATKO female mice exposed to 4°C for 10 days. (B) qPCR analysis of genes involved in glucose uptake, glycolysis, and FA oxidation in cold-activated IWAT (n=6/group). All data are presented as the Mean ± SEM. * p <0.05, ** p <0.01, **** p <0.0001.

Article Snippet: Lysates were separated by SDS-PAGE, transferred to nitrocellulose membrane, and analyzed by immunoblotting with following antibodies: GOT1 antibody (Pro Sci, #30-379), GOT2 antibody (Invitrogen, #PA5-27572), MDH1 antibody (Santa Cruz, #sc-166879), MDH2 antibody (Invitrogen, #PA5-21700), UCP1 antibody , GAPDH antibody (Abcam, #ab9485), VDAC antibody (Abcam, #ab15895), α-tubulin antibody (Abcam, #ab7291), β-actin antibody (Sigma, #A5441), phospho-PDH E1α antibody (S232) (Millipore, #AP106350).

Techniques: Staining

Journal: Journal of neurochemistry

Article Title: Synaptic vesicles are capable of synthesizing the VGLUT substrate glutamate from α-ketoglutarate for vesicular loading.

doi: 10.1111/j.1471-4159.2012.07684.x

Figure Lengend Snippet: Fig. 6 Vesicle-bound AAT belongs to the mitochondrial-bound AAT type and is associated with vesicles via ionic interaction. (a) Reactivity of vesicle-bound AAT and synsol AAT with antibodies against mito- chondrial AAT and cytosolic AAT. Synaptic vesicle fraction SV-A’ (4 lg) and the synsol fraction (4 lg) were subjected to SDS/western blotting, and each probed with antibodies against the cytosolic AAT (GOT1, 1 : 500 dilution) and the mitochondrial AAT (GOT2, 1 : 1000 dilution). (b) Vesicle-bound AAT is extracted with high salt. Synaptic vesicle fraction SV-A’ was treated with various concentrations of KCl and centrifuged, as described in Materials and methods, and super- natants (sup) and pellets (ppt) subjected to SDS/western blotting and probed with GOT2 antibody.

Article Snippet: Polyclonal antibodies to cytosolic and mitochondrial AAT (EC 2.6.1.1), referred to as GOT1 and GOT2 antibodies, respectively, were obtained from Aviva Systems Biology and Abnova, respectively.

Techniques: Western Blot

Journal: Communications Biology

Article Title: Glutamate oxaloacetate transaminase 1 is dispensable in macrophage differentiation and anti-pathogen response

doi: 10.1038/s42003-024-06479-w

Figure Lengend Snippet: a Schematic of AOAA treatment in BMDMs differentiated from Got1 f/f and Got1 ΔLysM mice. b , c Supernatant IL-6, TNFα protein levels of BMDMs derived from Got1 f/f and Got1 ΔLysM mice. BMDMs were pre-treated with 10 mM AOAA and then stimulated with 10 mM AOAA and LPS (20 ng/mL) + IFNγ (100 ng/mL) for 24 h. Got1 f/f mice, n = 3 biologically independent experiments, Got1 ΔLysM mice, n = 3 biologically independent experiments. d NO production level in BMDMs derived from Got1 f/f and Got1 ΔLysM mice. Seahorse analysis of OCR in M0 macrophages ( e ) and M1 macrophages ( f ), ECAR in M0 macrophages ( g ) and M1 macrophages ( h ). ns, p > 0.05, not significant, ** p < 0.01, *** p < 0.001, **** p < 0.0001, unpaired, p values were determined by two-tailed Student’s t test. Data are representative of three independent experiments (mean ± SD).

Article Snippet: Following blocking, the PVDF membrane was incubated overnight at 4°C with the primary antibody against GOT1 (1:1000 dilution, Abclonal A11363), GOT2 (1:1000 dilution, Abclonal A19245) and β-Actin (1:1000 dilution, Abcam ab8226).

Techniques: Derivative Assay, Two Tailed Test

Journal: Communications Biology

Article Title: Glutamate oxaloacetate transaminase 1 is dispensable in macrophage differentiation and anti-pathogen response

doi: 10.1038/s42003-024-06479-w

Figure Lengend Snippet: a Schematic of pro-inflammatory macrophages differentiation. b – d Il6 , Tnfa and Il1b mRNA levels in BMDMs stimulated with LPS (100 ng/mL) for 4 h or LPS (20 ng/mL) + IFNγ (100 ng/mL) for 24 h. Got1 f/f mice, n = 3 biologically independent experiments; Got1 ΔLysM mice, n = 3 biologically independent experiments. e , f IL-6, TNFα protein levels in the supernatant determined by ELISA. g NO production level in BMDMs stimulated with LPS (100 ng/mL) or LPS (20 ng/mL) + IFNγ (100 ng/mL) for 24 h. Got1 f/f mice, n = 3 biologically independent experiments; Got1 ΔLysM mice, n = 3 biologically independent experiments. h Lactate production levels in BMDMs stimulated with LPS (100 ng/mL) or LPS (20 ng/mL) + IFNγ (100 ng/mL) for 24 h. Got1 f/f mice, n = 3 biologically independent experiments; Got1 ΔLysM mice, n = 3 biologically independent experiments. ns, p > 0.05, not significant, unpaired, two-tailed Student’s t test. Data are representative of three independent experiments (mean ± SD).

Article Snippet: Following blocking, the PVDF membrane was incubated overnight at 4°C with the primary antibody against GOT1 (1:1000 dilution, Abclonal A11363), GOT2 (1:1000 dilution, Abclonal A19245) and β-Actin (1:1000 dilution, Abcam ab8226).

Techniques: Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Communications Biology

Article Title: Glutamate oxaloacetate transaminase 1 is dispensable in macrophage differentiation and anti-pathogen response

doi: 10.1038/s42003-024-06479-w

Figure Lengend Snippet: a Model of GOT1 in malate-aspartate shuttle. b Schematic of Aspartate treatment in wildtype BMDMs stimulated with LPS (100 ng/mL) or LPS (20 ng/mL) + IFNγ (100 ng/mL). c Il1b mRNA levels in BMDMs stimulated with LPS (100 ng/mL) for 4 h or LPS (20 ng/mL) + IFNγ (100 ng/mL) for 24 h. n = 3 biologically independent experiments. d – f Il6 , Tnfa and Il1b mRNA levels in BMDMs stimulated with LPS (20 ng/mL) + IFNγ (100 ng/mL) for 24 h. Got1 stop/+ mice, n = 3 biologically independent experiments; Got1 stop/+ ; Lyz2-Cre mice, n = 3 biologically independent experiments. g – i Il6 , Tnfa and Il1b mRNA levels in BMDMs stimulated with LPS (100 ng/mL) or HKCA (HKCA: BMDM = 1:1) for 4 h. Got1 stop/+ mice, n = 3 biologically independent experiments; Got1 stop/+ ; Lyz2-Cre mice, n = 3 biologically independent experiments. j , k IL-6, TNFα protein levels in the supernatant determined by ELISA. BMDMs were stimulated with 100 ng/mL LPS, LPS (20 ng/mL) + IFNγ (100 ng/mL) or HKCA (HKCA: BMDM = 1:1) for 24 h. Got1 f/f mice, n = 3 biologically independent experiments; Got1 ΔLysM mice, n = 3 biologically independent experiments. ns, p > 0.05, not significant, * p < 0.05, ** p < 0.01, unpaired, two-tailed Student’s t test. Data are representative of three independent experiments (mean ± SD).

Article Snippet: Following blocking, the PVDF membrane was incubated overnight at 4°C with the primary antibody against GOT1 (1:1000 dilution, Abclonal A11363), GOT2 (1:1000 dilution, Abclonal A19245) and β-Actin (1:1000 dilution, Abcam ab8226).

Techniques: Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Communications Biology

Article Title: Glutamate oxaloacetate transaminase 1 is dispensable in macrophage differentiation and anti-pathogen response

doi: 10.1038/s42003-024-06479-w

Figure Lengend Snippet: a Schematic of ROS detection in macrophages. b ROS production in BMDMs. Prior to ROS detection, BMDMs were pre-treated with AOAA for 24 h, then treated with HKCA (HKCA: BMDM = 1:1) for 1 h. Got1 f/f mice, n = 3 biologically independent experiments; Got1 ΔLysM mice, n = 3 biologically independent experiments. c Phagocytosis of BMDMs. FITC labeled-HKCA was utilized for phagocytosis assay with BMDMs. d CFU Count of S. aureus and E. coli in BMDMs-In-Vitro-Killing Assay. e Schematic of infection design. f , g Percentage of mice survival. ns, p > 0.05, not significant, * p < 0.05, ** p < 0.01, unpaired, p values were determined by two-tailed Student’s t test in b – d and log-rank test was used for survival comparison in f and g . Data are representative of three independent experiments (mean ± SD).

Article Snippet: Following blocking, the PVDF membrane was incubated overnight at 4°C with the primary antibody against GOT1 (1:1000 dilution, Abclonal A11363), GOT2 (1:1000 dilution, Abclonal A19245) and β-Actin (1:1000 dilution, Abcam ab8226).

Techniques: Labeling, Phagocytosis Assay, In Vitro, Infection, Two Tailed Test, Comparison

Journal: Communications Biology

Article Title: Glutamate oxaloacetate transaminase 1 is dispensable in macrophage differentiation and anti-pathogen response

doi: 10.1038/s42003-024-06479-w

Figure Lengend Snippet: a Schematic of the differentiation of M2 macrophages. b – d Chil3 , Arg1 , and Retnla mRNA levels in BMDMs, derived from Got1 f/f and Got1 ΔLysM mice, pretreated with 10 mM AOAA for 24 h, then treated with IL-4 (20 ng/mL) for 24 h. n = 3 biologically independent experiments. e – g Chil3 , Arg1 , and Retnla mRNA levels in PMs, derived from Got1 f/f and Got1 ΔLysM mice, pretreated with 10 mM AOAA for 24 h, then treated with IL-4 (20 ng/mL) for 24 h. n = 3 biologically independent experiments. h – j Chil3 , Arg1 , and Retnla mRNA levels in BMDMs, derived from Got1 stop/+ and Got1 stop/+ ; Lyz2-Cre mice, treated with IL-4 (20 ng/mL) for 24 h. n = 3 biologically independent experiments. ns, p > 0.05, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, unpaired, p values were determined by two-tailed Student’s t test. Data are representative of three independent experiments (mean ± SD).

Article Snippet: Following blocking, the PVDF membrane was incubated overnight at 4°C with the primary antibody against GOT1 (1:1000 dilution, Abclonal A11363), GOT2 (1:1000 dilution, Abclonal A19245) and β-Actin (1:1000 dilution, Abcam ab8226).

Techniques: Derivative Assay, Two Tailed Test

Journal: Communications Biology

Article Title: Glutamate oxaloacetate transaminase 1 is dispensable in macrophage differentiation and anti-pathogen response

doi: 10.1038/s42003-024-06479-w

Figure Lengend Snippet: a Schematic of immune tolerance induced with LPS. b , c IL-6, TNFα protein levels in the supernatant of Got1 f/f and Got1 ΔLysM mice derived BMDMs were determined by ELISA. Before LPS retreatment, BMDMs were pretreated with 10 mM AOAA for 24 h, n = 3 biologically independent experiments. d Lactate production level in BMDMs stimulated with LPS (100 ng/mL) retreatment for 24 h. e NO production level in BMDMs stimulated with LPS (100 ng/mL) retreatment for 24 h. f , g IL-6, TNFα protein levels in the supernatant of Got1 stop/+ and Got1 stop/+ ; Lyz2-Cre mice derived BMDMs were determined by ELISA in. n = 3 biologically independent experiments. ns, p > 0.05, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, unpaired, p values were determined by two-tailed Student’s t test. Data are representative of three independent experiments (mean ± SD).

Article Snippet: Following blocking, the PVDF membrane was incubated overnight at 4°C with the primary antibody against GOT1 (1:1000 dilution, Abclonal A11363), GOT2 (1:1000 dilution, Abclonal A19245) and β-Actin (1:1000 dilution, Abcam ab8226).

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Cancer & Metabolism

Article Title: PGC-1α supports glutamine metabolism in breast cancer

doi: 10.1186/2049-3002-1-22

Figure Lengend Snippet: PGC-1α modulates the expression of glutamine metabolism enzymes. (A) Schematic representation of glutamine metabolism in cells. SLC1A5, solute carrier family 1, member 5; GLS1, kidney-type glutaminase; GLS2, liver-type glutaminase; GLUD1, glutamate dehydrogenase 1; GOT2, mitochondrial glutamic-oxaloacetic transaminase; GPT2, mitochondrial glutamic pyruvate transaminase; GOT1, cytoplasmic glutamic-oxaloacetic transaminase; GPT1, cytoplasmic glutamic pyruvate transaminase; GLUL, glutamate-ammonia ligase; Gln, glutamine; Glu, glutamate; OAA, oxaloacetate; Asp, aspartate; Pyr, pyruvate; Ala, alanine; α-KG, α-ketoglutarate; CAC, citric acid cycle. (B) Expression of glutamine metabolism genes in ERBB2/Neu-induced breast cancer cells (NT2196) with increased expression of PGC-1α (α-1.1, α-1.2) normalized to that in control cells. Data are presented as means ± S.E.M., n = 4. * P <0.05, paired Student's t -test. (C) ChIP analyses for PGC-1α and ERRα at ERRE sites on the promoter of glutamine metabolism genes in NT2196 cells with increased expression of PGC-1α (α-1.1). Data represent the fold enrichment for two independent experiments. (D) Representative western blot for select glutamine metabolism enzymes in NT2196 cells with increased expression of PGC-1α (α-1.1, α-1.2) and control (Ctl-1). (E) Quantification of five independent western blot experiments, normalized to ACTIN levels. Data are presented as means ± S.E.M., n = 5. * P <0.05, paired Student's t -test.

Article Snippet: The blots were incubated according to the manufacturer's instructions with the following primary antibodies: Glud1 (GeneTex, Irvine, CA, USA, GTX88164), Gls (Abcam, Toronto, Canada, AB93434), Got1 (GeneTex, GTX88903), Got2 (GeneTex, GTX88925), Hsp90 (Santa Cruz Biotechnology, sc-101494), and Actin (Santa Cruz Biotechnology, sc-1616) and with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare, Mississauga, Canada, or Santa Cruz Biotechnology).

Techniques: Expressing, Control, Western Blot