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: Cell reports

Article Title: Respiratory Supercomplexes Promote Mitochondrial Efficiency and Growth in Severely Hypoxic Pancreatic Cancer

doi: 10.1016/j.celrep.2020.108231

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal anti-GOT2 , Novus Biologicals , Cat#NBPI-47469; Lot#160630; RRID:AB_10010421.

Techniques: Plasmid Preparation, Virus, Recombinant, Clinical Proteomics, Membrane, Protease Inhibitor, Avidin-Biotin Assay, Fractionation, Software

Journal: Nature Communications

Article Title: Metabolic profiling reveals channeled de novo pyrimidine and purine biosynthesis fueled by mitochondrially generated aspartic acid in cancer cells

doi: 10.1038/s41467-025-64013-w

Figure Lengend Snippet: A Schematic describing glutamine (Gln) mediated 15 N label incorporation in pyrimidine de novo biosynthesis intermediates and end product UMP (also see schematic Fig. ). Various isotope labeled analytes generated during the course of the assay are: 15 N2 Gln (sidechain amide nitrogen: open red circle, backbone amine nitrogen: solid red circle), 15 N glutamic acid (Glu, backbone amine nitrogen: solid red circle), and 15 N aspartic acid (Asp, backbone amine nitrogen: solid red circle). The pyrimidine nucleotide UMP (N 1 and N 3 ) with the labeled positions arising from Gln side chain and Asp backbone, respectively, are marked on the representative pyrimidine ring. The enzyme glutamate oxaloacetate transaminase GOT1 (cytosolic) or GOT2 (mitochondria) catalyzes the conversion of oxaloacetic acid (OAA) to Asp, transferring the backbone amine of Glu to Asp. Two alternative models for pyrimidine de novo biosynthesis: B completely diffusive versus C channeled pathway with CAD and UMPS coordinating with DHODH, respectively. C A channeled mechanism-based model for the pyrimidine de novo biosynthesis metabolon, the pyrimidinosome (blue background), with CAD and UMPS that assemble proximal to mitochondria and DHODH , allowing preferential utilization of the mitochondrially generated 15 N Asp. In this model, mitochondrial GOT2 serves as the primary source of 15 N Asp utilized for channeled pyrimidine biosynthesis. Diffusive model predicts homogenous 15 N isotope label incorporation across the various substrates and product, while the channeled pathway predicts isotope label inhomogeneity. Enzyme names are italicized and Asp, Gln mitochondrial transporters are shown in magenta and green, respectively. To test which model applies to pyrimidine synthesis, 15 N2 Gln labeling was carried out for 2 h. The % 15 N labeled Asp incorporation in the analytes of interest is shown: D UDP and Asp and E carbamoyl aspartate, dihydroorotate, and orotate. The % 15 N labeled Asp incorporation was calculated as = \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left(M+1.994\right)\div\left[\left(M+.997\right)+\left(M+1.994\right)\right]$$\end{document} M + 1.994 ÷ M + . 997 + M + 1.994 . Data from three independent biological replicates are shown, bar heights represent the mean. ** p -value < 0.005 for a pairwise comparison using two-way t-test (Additional data in supplementary Fig. ). A , B , and C prepared using Biorender and Chemdraw structures were used in A https://BioRender.com/x25177d .

Article Snippet: Anti-GOT2 primary antibody (rabbit polyclonal anti-GOT2 antibody, #NBP1-81565, Novus Biologicals, Inc.), anti-pyruvate carboxylase (rabbit anti-PC, #NBP1-49536SS, Novus Biologicals, Inc.) anti-GOT1 (rabbit polyclonal anti-GOT1, #PA5-80670 Invitrogen) anti-PFAS primary antibody (rabbit anti-PFAS #A304-218A, Bethyl Labs), anti-α-tubulin (#DM1A, Cell Signaling), or anti-β-actin antibody (mouse anti-β-actin #10R-2927, Fitzgerald) were reconstituted in the blocking buffer at 1:2000 dilution and overnight staining was allowed after which the membrane was washed thrice with TBST buffer thrice at RT.

Techniques: Labeling, Generated, Transferring, Comparison

Journal: Nature Communications

Article Title: Metabolic profiling reveals channeled de novo pyrimidine and purine biosynthesis fueled by mitochondrially generated aspartic acid in cancer cells

doi: 10.1038/s41467-025-64013-w

Figure Lengend Snippet: A Schematic describing the effect of antimycin A, an electron transport chain Complex-III (C-III) inhibitor. Antimycin A treatment increases FADH 2 /FADH, NADH/NAD ratios, thus, B increasing succinate/fumarate ratio and inhibiting the TCA cycle, while the activity of PC goes unperturbed. PC activity only generates (M + 3) isotopologue due to lack of 13 C CO 2 upon TCA cycle inhibition. C The isotopologue distribution in succinate D aspartate and E OAA after 6 h 13 C6 Glc incorporation following antimycin A treatment of HeLa cells (Additional data in Fig. ). F Schematic showing the involvement of mitochondrial PC in generation of (M + 3) and (M + 4) isotopologues of Asp and (M + 7) and (M + 8) isotopologues of UTP. An effect on the fractional abundance of (M + 8) UTP is expected, since this isotopologue is uniquely generated by PC activity at 6 h labeling time point. Both TCA cycle as well as PC activity contribute to the (M + 7) isotopologue. G Isotopologue distribution in UTP after 6 h 13 C6 Glc incorporation following 72 h treatment with the control non-target (NTsi) or the pyruvate carboxylase (PCsi) siRNA. (Additional data in Fig. ). H Comparison of the observed (M + 6), (M + 7), and (M + 8) isotopologue distribution in UTP after 6 and 2 h of 13 C6 Glc mediated label incorporation. I Fractional abundance of the (M + 8) isotopologue in UTP following the indicated 13 C6 Glc labeling times (2–8 h) (Additional data in Fig. ). J Diffusive model-based prediction (using observed orotate isotopologue abundance) and observed isotopologue distribution in UTP after 14 h 13 C6 Glc label incorporation (Additional data in Fig. ). K Schematic showing the metabolic effect of GOT2 knockdown. Owing to the role of mitochondrial GOT2 in supporting the channeled pyrimidine de novo synthesis, its knockdown rewires the mitochondrial and cytosolic metabolism as shown in the schematic, thus perturbing the channeled flux, while allowing the accumulation of intermediates generated by diffusive mechanism. L The ratio of total peak area of the substrate Asp to the pyrimidine nucleotide UMP in GOT2 knockdown cells compared to the control non-target siRNA treated cells. M The ratio of total peak area of the pyrimidine biosynthesis intermediates (orotate and dihydroorotate, DHO) to UMP in GOT2 knockdown cells compared to the control non-target siRNA treated cells (Additional data in Fig. ). N Schematic showing the link between the cytosolic GOT1 activity and the mitochondrial malate-aspartate shuttle (MAS), which supports the TCA cycle. Various enzymes involved in the shuttle ensure proton gradient across the mitochondrial membrane and support the directional flow of electron and protons across the electron transport chain (ETC), which in turn supports DHODH activity by generating oxidized CoQ. O and P the ratios of carbamoyl aspartate (CA) to orotate and DHO to orotate, respectively, in antimycin A treated cells. Q and R the ratios of CA to orotate and DHO to orotate, respectively, after GOT1 knockdown compared to that in the control non-target siRNA treated HeLa cells. S The observed (M + 6), (M + 7), and (M + 8) isotopologue distribution in UTP after 6 h of 13 C6 Glc following GOT1 knockdown (Additional data in Fig. ). B , C , D , E , H , I , J , O , P , Q , R , and S data from three or in ( G ), ( L ), and ( M ) data from four independent biological replicates are shown, bar heights represent data mean. Pairwise ratio comparison using two-way t-test was performed in ( J and S ), unpaired two-way t-test was performed for ( G ), ( H ), ( L ), ( M ), ( O ), ( P ), ( Q ), ( R ), and one way ANOVA was performed in ( I ). Pathway steps impacted by drug or siRNA treatment are indicated as red inhibition arrow. A , F , K , and N prepared in Biorender https://BioRender.com/x25177d .

Article Snippet: Anti-GOT2 primary antibody (rabbit polyclonal anti-GOT2 antibody, #NBP1-81565, Novus Biologicals, Inc.), anti-pyruvate carboxylase (rabbit anti-PC, #NBP1-49536SS, Novus Biologicals, Inc.) anti-GOT1 (rabbit polyclonal anti-GOT1, #PA5-80670 Invitrogen) anti-PFAS primary antibody (rabbit anti-PFAS #A304-218A, Bethyl Labs), anti-α-tubulin (#DM1A, Cell Signaling), or anti-β-actin antibody (mouse anti-β-actin #10R-2927, Fitzgerald) were reconstituted in the blocking buffer at 1:2000 dilution and overnight staining was allowed after which the membrane was washed thrice with TBST buffer thrice at RT.

Techniques: Activity Assay, Inhibition, Generated, Labeling, Control, Comparison, Knockdown, Membrane

Journal: Nature Communications

Article Title: Metabolic profiling reveals channeled de novo pyrimidine and purine biosynthesis fueled by mitochondrially generated aspartic acid in cancer cells

doi: 10.1038/s41467-025-64013-w

Figure Lengend Snippet: A Schematic describing the incorporation of 15 N side chain amide nitrogen of Gln (red open circles) and backbone amine nitrogen of Asp (red solid circles) into the purine ring following diffusive de novo purine biosynthesis. The first two 15 N incorporations are catalyzed by the enzymes PPAT and PFAS (utilizing Gln) and the third and fourth 15 N incorporations are catalyzed by the enzymes PAICS and ADSS (utilizing Asp), respectively. Note that following the hypothesis that the diffusive and the channeled DNPB utilize the same source of 15 N Asp, the respective fraction 15 N Asp (x) incorporation in SAICAR and AMP will be expected to be the same, thus allowing estimation of the relative abundances of the (M + 2), (M + 3), and (M + 4) isotopologues in AMP based on the value of (x) obtained from SAICAR. When the % 15 N Gln incorporation in the intermediates is ~100%, the (M + 2), (M + 3), and (M + 4) isotopologues in AMP show no interference from the preexisting unlabeled nucleotide pools of AMP. Fraction Isotopologue abundance observed in the DNPB intermediates B FGAR, C SAICAR, and D ADP, respectively (Additional data in Fig. ). D Based on the fraction 15 N Asp (x) in SAICAR, the respective fractional abundance of (M + 2), (M + 3), and (M + 4) isotopologues in ATP was predicted based on the diffusive model (Eq5 Supplementary Information) and compared to the observed values. E The observed fraction 15 N Asp incorporation in SAICAR and ADP, respectively. F Schematic showing the preferential 15 N incorporation via Gln and Asp into the purine ring following channeled de novo purine synthesis by mitochondria-proximal purinosomes. Purinosome metabolon formation leads to channeled synthesis by restricting the diffusion of the intermediates, only allowing the end nucleotides AMP and GMP to be released in the bulk cytosol. G Schematic showing the effect of GOT2 knockdown on channeled purine synthesis. Pathway steps impacted by siRNA treatment are indicated as red inhibition arrows. As the production of Asp in mitochondria is suppressed, GOT1 mediated cytoplasmic production increases, supporting diffusive purine de novo biosynthesis, leading to accumulation of both Asp as well as the purine biosynthesis intermediates. H The relative ratio of total peak area of the substrate Asp to the purine nucleotides AMP and GMP, respectively, in GOT2 knockdown cells compared to the control non-target siRNA treated cells. I The ratio of total peak area of purine biosynthesis intermediate IMP to the end product nucleotides AMP and GMP, respectively, in GOT2 knockdown cells compared to the control non-target siRNA treated cells. (Associated supporting data in Figs. D, and ). B , C , D , E data from three or H , and I four independent biological replicates are shown, bar height represents data mean. * p -value < 0.05, ** p -value < 0.005, *** p -value < 0.001 for a ( D ), ( E ) pairwise or ( H ), ( I ) unpaired two-ways t-test. A , F , and G prepared in Biorender https://BioRender.com/x25177d .

Article Snippet: Anti-GOT2 primary antibody (rabbit polyclonal anti-GOT2 antibody, #NBP1-81565, Novus Biologicals, Inc.), anti-pyruvate carboxylase (rabbit anti-PC, #NBP1-49536SS, Novus Biologicals, Inc.) anti-GOT1 (rabbit polyclonal anti-GOT1, #PA5-80670 Invitrogen) anti-PFAS primary antibody (rabbit anti-PFAS #A304-218A, Bethyl Labs), anti-α-tubulin (#DM1A, Cell Signaling), or anti-β-actin antibody (mouse anti-β-actin #10R-2927, Fitzgerald) were reconstituted in the blocking buffer at 1:2000 dilution and overnight staining was allowed after which the membrane was washed thrice with TBST buffer thrice at RT.

Techniques: Diffusion-based Assay, Knockdown, Inhibition, Control

Journal: PLoS ONE

Article Title: Febuxostat ameliorates secondary progressive experimental autoimmune encephalomyelitis by restoring mitochondrial energy production in a GOT2-dependent manner

doi: 10.1371/journal.pone.0187215

Figure Lengend Snippet: (A) Representative alterations in gene expression detected by DNA microarray analysis in brains and spinal cords from mice with secondary progressive experimental autoimmune encephalomyelitis (SP-EAE) that were treated with febuxostat (Fx) or vehicle. Pink represents upregulation, while blue represents those downregulation of genes. (B) Schematic of an alternative pathway for ATP synthesis associated with GOT2 and the Krebs cycle. (C) RT-PCR analysis of GOT2 gene expression in spinal cords isolated from naïve, SP-EAE mice and SP-EAE mice treated with Fx (naïve; n = 3, EAE; n = 3, EAE+Fx; n = 3). (D) Western blot analysis of GOT2 protein expression in spinal cords isolated from naïve mice and SP-EAE mice treated with vehicle or Fx. β-actin was used as an internal control. (E) The relative expression level of GOT2 to β-actin for each depicted group (naïve, n = 3; SP-EAE, n = 3; SP-EAE+Fx, n = 3). (F) Immunohistochemical analysis of GOT2 expression in the lumbar cords isolated from naïve mice, SP-EAE mice or SP-EAE mice treated with Fx. Lines indicate borders between white matter and gray matter. Hematoxylin was used as a counterstain. The magnified image illustrates the dot-like expression of GOT2 in neuronal somata. (G) Immunofluorescent staining analysis of GOT2 and MAP2 or TOM20 in lumbar cords isolated from naïve mice indicates GOT2 is expressed in neuronal mitochondria.

Article Snippet: The primary antibodies used were a rabbit monoclonal antibody against GOT2 (1:50; Sigma-Aldrich) or mouse monoclonal antibody against GOT2 (1:100; Abcam) and then incubated over-night at 4°C.

Techniques: Expressing, Microarray, Reverse Transcription Polymerase Chain Reaction, Isolation, Western Blot, Immunohistochemical staining, Staining

Journal: PLoS ONE

Article Title: Febuxostat ameliorates secondary progressive experimental autoimmune encephalomyelitis by restoring mitochondrial energy production in a GOT2-dependent manner

doi: 10.1371/journal.pone.0187215

Figure Lengend Snippet: (A, B) Neuro2a cells were treated with 10 μM of Fx or vehicle (DMSO) for 24 h and the expression level of GOT2 was assessed by RT-PCR (A) and immunofluorescence (B). Data are mean ± SEM of triplicate cultures (A). * indicates P ≤ 0.05. One-way analysis of Student T-tests was used to compare two groups. (C) GOT2 (red) was colocalized with TOM20-positive mitochondria (green).

Article Snippet: The primary antibodies used were a rabbit monoclonal antibody against GOT2 (1:50; Sigma-Aldrich) or mouse monoclonal antibody against GOT2 (1:100; Abcam) and then incubated over-night at 4°C.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence

Journal: PLoS ONE

Article Title: Febuxostat ameliorates secondary progressive experimental autoimmune encephalomyelitis by restoring mitochondrial energy production in a GOT2-dependent manner

doi: 10.1371/journal.pone.0187215

Figure Lengend Snippet: Neuro2a cells were transfected with control siRNA or GOT2 siRNA. RT-PCR analysis (A) and western blot analysis (B) confirmed the silencing of GOT2 gene and protein expression, respectively. (C) ATP production was assessed in Neuro2a cells that were transfected either with control or GOT2-specific siRNA and treated with DMSO or 10 μM of Fx for 12 h. (D) Neuro2a cells that were transfected with either control or GOT2-specific siRNA were stimulated with 10 nM of rotenone (Rt, 6 h) after pre-treatment with DMSO or 10 μM of Fx for 12 h. Data are mean ± SEM of triplicate cultures. Error bars indicate standard error. All data are representative of 3 independent experiments. * indicates P ≤ 0.05. One-way analysis of Student T-tests was used to compare two groups and a one-way analysis of variance (ANOVA) was used to compare more than two groups.

Article Snippet: The primary antibodies used were a rabbit monoclonal antibody against GOT2 (1:50; Sigma-Aldrich) or mouse monoclonal antibody against GOT2 (1:100; Abcam) and then incubated over-night at 4°C.

Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing