459400

antibody from Invitrogen Antibodies

Targeting: PDHA1 PDHA

Provider product page for 459400

Western blot

Immunocytochemistry

Flow cytometry

Other assay

Antibody data

Antibody Data
Antigen structure
References [17]
Comments [0]
Validations
Western blot [2]
Immunocytochemistry [1]
Other assay [11]

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Product number: 459400 - Provider product page
Provider: Invitrogen Antibodies
Product name: PDHA1 Monoclonal Antibody (9H9AF5)
Antibody type: Monoclonal
Antigen: Recombinant full-length protein
Reactivity: Human, Mouse, Rat, Bovine
Host: Mouse
Isotype: IgG
Antibody clone number: 9H9AF5
Vial size: 100 µg
Concentration: 1 mg/mL
Storage: 4° C, do not freeze

PDHA1 protein structure - 459400 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of PDH was achieved by transfecting HEK-293 cells with PDH specific siRNAs (Silencer® select Product # s10244). Western blot analysis (Fig. a) was performed using whole cell extracts from PDH knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blot was probed with PDHA1 Monoclonal Antibody (9H9AF5) (Product # 459400, 1µg/ml) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 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 PDH.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-PDHA1 Monoclonal Antibody (9H9AF5) (Product # 459400) and a band at ~43 kDa corresponding to PDH was observed in all the cell lines and tissues tested. Whole cell extracts (30 µg lysate) of Hep G2 (Lane 1), HEK 293 (Lane 2), MDA-MB-231 (Lane 3), HL-60 (Lane 4), SK-OV-3 (Lane 5), PC-3 (Lane 6), PANC-1 (Lane 7), Mouse Liver (Lane 8), Rat Liver (Lane 9), Mouse Brown Adipose (Lane 10), Mouse Brain (Lane 11) were electrophoresed using Novex® NuPAGE® 4-12% % Bis-Tris gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1µg/ml) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005)..

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 Dichloroacetate treatment reduced phosphorylation of PDHE1 alpha at pSer 293 site with no effect on the levels of total PDHE1 alpha in all the cell lines investigated. Whole-cell lysates were prepared after treating cells with 20 m M DCA for 8 h and from untreated cells, and western blot analyses were performed.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Mutant Kras copy-number dictates redox state, metabolic dependencies and therapeutic susceptibilities a , Total and phosphorylated Pdhe1a levels and Pdh activity in MEFs. b, Cellular ROS (CellRox); c, NADPH/NADP + ratio and NADPH levels; d, GSH/GSSG ratio and GSH levels in MEFs. e, MEF survival upon 24 hrs H 2 O 2 treatment. f, Nrf2 and Nrf2-target gene expression in MEFs (left: qPCR; right: microarray). Nrf2-targets significantly upregulated in homozygous MEFs highlighted (bold red, t-test). g, MEF viability after 72 hrs culture in low glucose (Low GLC), 2DG or low glutamine (Low GLN), relative to normal media (CTRL). h, Percentage of AnnexinV + /PI + (AnV/PI positive, FACS) MEFs upon 48 hrs BSO, 2DG, or combined (2DG+BSO) treatment. i, GSH/GSSG ratio and GSH levels in KRAS mut NSCLC cells. j, NSCLC cells treated as in (h) . Triplicate mean +-s.d. shown for three independent MEFs/genotype ( a,c,d,f ) or for representative data (3 independent runs) ( b,e,g,h,i,j ). Data normalised to WT/WT ( a,c-f ) or HET mean ( i ). One-way ( a-f,i ) or two-way ANOVA ( g,h,j). *** P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1 Reductive glutamine metabolism in spheroids a , Mass isotopolgue analysis of citrate in H460 cells cultured with [U- 13 C]glucose and unlabeled glutamine (n=3 cultures from a representative experiment). b , Oxygen consumption rates (OCR) of cells grown in monolayer or spheroid culture (n=10 monolayer cultures and 11 spheroids from a representative experiment). c , Western blot for total (t) and phosphorylated (p, Ser293) PDH, and PDH kinase-1 (PDK1). d , Mass isotopologue analysis of citrate in cells cultured with [U- 13 C]glutamine and unlabeled glucose (n=3 cultures from a representative experiment). e , Evolution of citrate mass isotopologues in spheroids cultured with [U- 13 C]glutamine (n=2 cultures for each time point). f , Citrate m+4 and m+5 isotopologues in monolayer and spheroid cultures of A549, HT-29 and MCF7 cells cultured with [U- 13 C]glutamine (n=3 A549 monolayer cultures; n=4 cultures for all other conditions). Complete mass isotopologue distributions are in Supplementary Table 1 . *p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 Pdha1 knockout induces tumour suppression in mice and human prostate tumours. ( a ) Western blot analysis of indicated proteins in wild type, Pdha1 pc-/Y , Pten pc-/- and Pten pc-/- ; Pdha1 pc-/Y prostates and tumours (n=3, independent prostate samples). Uncropped images are in Supplementary Figure 12 . ( b ) Upper panel, PDC activity measurement in wild type, Pdha1 pc-/Y , Pten pc-/- and Pten pc-/- ; Pdha1 pc-/Y prostates and tumours (n=3, independent prostate samples). Lower panel, Quantification of indicated proteins normalized to wild type tissues in indicated prostate tumours in ( a ) (n=3, independent prostate samples). ( c ) Comparison of anterior prostate (AP) lobe volumes (mm 3 , 2 independent lobes per animal are presented) from male of indicated ages and genotypes between wild type, Pdha1 pc-/Y , Pten pc-/- and Pten pc-/- ; Pdha1 pc-/Y prostate and tumours (N, the number of mice of indicated ages). Inset is the representative image of anterior prostate lobes in the panel. ( d ) Representative micrographs in histopathological analysis (haematoxylin/eosin staining and indicated proteins) of anterior prostates (AP) in Pdha1 pc-/Y , Pten pc-/- and Pten pc-/- ; Pdha1 pc-/Y prostate tissues from 12 weeks old male mice (n=3) (Scale bar represents 50 mum, insets are regions shown in higher magnification, see also Supplementary Fig. 1c for all the genotypes and images of lower magnification). ( e,f ) Quantification of the percentage of Ki-67 positive cells ( e ) an

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Pdha1 inactivation induces tumour suppression by down-regulating lipogenic genes. ( a ) Gene expression profile analysis based on metabolic pathway datasets (GOCC, Gene Ontology Cellular Component; KEGG, Kyoto Encyclopaedia of Genes and Genomes; GOBO, Gene expression-based Outcome; GOMF, Gene Ontology Molecular Function; HumanCyc; Reactome) in Pten pc-/- ; Pdha1 pc-/Y tumours versus Pten pc-/- tumours. Dotted line indicates P =0.05 (n=3). ( b ) Western blot analysis of indicated proteins in wild type, Pdha1 pc-/Y , Pten pc-/- and Pten pc-/- ; Pdha1 pc-/Y prostates and tumours. Uncropped images are in Supplementary Figure 12 . (n=3, independent prostate samples). ( c ) Western blot analysis of indicated proteins in 22Rv1 cells infected with doxycycline-induced Tripz-sh PDHA1 or scramble control and treated with 100 muM acetate or vehicle over a 6-day period. Uncropped images are in Supplementary Figure 12 . (n=3, independent cell cultures). ( d ) Upper panel, GSEA of SREBF target genes in Pten pc-/- ; Pdha1 pc-/Y versus Pten pc-/- prostate tumours. Normalized enriched scores (NES) are presented. Data are mean +- standard deviation (s.d.). Lower panel, quantitative real time-PCR analysis of mRNA expression of indicated SREBFs and target genes and genes in acetyl CoA compensatory pathways in mouse prostate and tumours the indicated genotypes (n=3). ( e ) Quantitative real time-PCR analysis of mRNA expression of indicated SREBFs and target genes and genes in acetyl coA c

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 Nuclear PDC regulates the expression of lipid biosynthesis genes independently of mitochondrial PDC. ( a ) Western blot analysis of indicated proteins in nuclear and cytoplasmic fractions of sh PDHA1 22Rv1 cells infected with NES-PDHA1 and NLS-PDHA1 alone or in combination. Uncropped images are in Supplementary Figure 13 . (n=3, independent cell cultures). ( b ) Western blot analysis of indicated proteins in sh PDHA1 22Rv1 cells infected NES-PDHA1 and NLS-PDHA1 alone or in combination (see full panel in Supplementary Fig. 5b ). Uncropped images are in Supplementary Figure 13 . (n=3, independent cell cultures). ( c-e ) Quantitative real-time PCR analysis of mRNA expression for ACLY ( c ) and SQLE ( d ) and determination of citrate levels ( e ) in shRNA control and sh PDHA1 22Rv1 and PC3 infected with NES-PDHA1, NLS-PDHA1 alone or in combination (n=3, independent cell cultures). ( f ) Representative confocal images and quantification of lipid droplets (average lipid droplets per cell) in sh PDHA1 22Rv1 infected with NES-PDHA1, NLS-PDHA1 alone or in combination (n=3, independent cell cultures, Scale bar represents 10 um, 5 fields acquired for each group). ( g ) Upper panel, representative images of crystal violet staining of sh PDHA1 22Rv1 infected with infected with NES-PDHA1, NLS-PDHA1 alone or in combination (n=3, independent cell cultures). Lower panel, relative cell number quantification by crystal violet staining in shRNA control and sh PDHA1 22Rv1 and PC3 infecte

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5 Nuclear PDC regulates fatty acid synthesis in presence of mitochondrial citrate. ( a,b ) Relative cell number quantification by crystal violet ( a, also see full panel in Supplementary Fig. 8a ) and quantification by confocal microscopy of average lipid droplets per cell ( b ) in sh PDHA1 22Rv1 and PC3 cells infected with NES-PDHA1, NLS-PDHA1 alone or in combination and treated with citrate (100 uM) or vehicle for 6 days (n=3, independent cell cultures). ( c ) Upper panel, Representative confocal images, and quantification of average lipid droplets per cell, in xenograft tumours from shRNA control and sh PDHA1 22Rv1 cells infected with PDK1 or empty vector. Lower panel, Evaluation of tumour formation in xenotransplantation experiments in shRNA control and sh PDHA1 22Rv1 cells infected with PDK1 or empty vector (n=6 animals; 12 injections, 5 fields acquired for each group and Scale Bar represents 20 mum). ( d ) Upper panel, representative immune-histochemistry micrographs for Ki-67 staining in tumours of the indicated genotypes. (n=6 animals; 12 injections, 5 fields acquired for each group and scale bar represents 50 mum). Lower panel, quantification of the percentage of Ki-67 positive cells in different tumour genotypes (n=6 animals; 12 injections, 5 fields acquired for each group). ( e,f ) Determination of citrate levels ( e ) and western blot analysis of indicated proteins ( f ) in xenograft tumours from shRNA control and sh PDHA1 22Rv1 cells infected with PDK1 or