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  • 86
    Santa Cruz Biotechnology gabpa
    AMPK regulates <t>HIF1α</t> and <t>GABPA</t> through CREB1 a , ChIP using CREB1 antibody and quantitative PCR showing CREB1 binding to promoter/enhancer regions of HIF1α and GABPA. (n = 3). *p = 0.009; + 0.005. b , Quantification of HIF1α promoter activity in control (NT) and CREB1-silenced GSCs expressing HRE-firefly and renilla luciferase reporter plasmids. (n = 3). * p = 0.01. + 0.008. c , Q-RTPCR analysis of GSCs expressing NT or CREB1 shRNA. (n = 3). *p = 0.002; + 0.001; # 0.004; $ 0.007; **0.01; ++ 0.0005. d , Viability of GSCs expressing NT or CREB1 shRNA. (n = 3). *p = 0.003; + 0.01. Inset: WB of CREB1. e, f , ECAR and OCR in GSCs expressing CREB1 shRNA. (n = 3). *p = 0.01; + 0.006. g, h , WB of pCREB1 S133 and CREB1 (loading control) in GSCs expressing NTshRNA or two independent AMPKβ1shRNA. i , WB of pACC, pCREB1 and CREB1 in normal human astrocytes (NHA) treated with AMPK activator A769662 or glucose starvation (30 min). j , IHC of pCREB S133 in tumors derived from GSCs expressing NT or AMPKβ1 shRNA. Scale bar 100µm. k , Q-RTPCR analysis of GSC 326 expressing control virus, CREB1 S133A , or CREB1 S133E lentivirus. (n = 3). *p = 0.01; + ≤ 0.0003; $ 0.002; # 0.02; **0.03. I , WB of HIF1α and GABPA in GSCs expressing CREB1 shRNA, CREB1 S133A and CREB1 S133E mutants. Total CREB1 and actin are also shown. m , WB of HIF1α and GABPA in tumors derived from GSCs expressing S133A .
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    94
    Santa Cruz Biotechnology anti gabpa
    <t>GABPA</t> expression is higher in the luminal subtype and associated with luminal signature, and predicts patient survival in bladder cancer (BC). a Schematic diagram showing luminal and basal BCs and their gene expression signatures. b Differential GABPA expression between luminal and basal subtypes of BCs. The TCGA cluster I (luminal) and III (basal) were compared for their GABPA and TERT (Telomerase reverse transcriptase) mRNA expression. Their mRNA levels are expressed as transcript reads determined using RNA sequencing. TERT telomerase reverse transcriptase. c Positive correlation between GABPA and <t>FoxA1/GATA3</t> mRNA expression in BCs. The TCGA and GSE32894 cohorts of BC patients were analyzed for the relationship of GABPA with FoxA1 and GATA3 transcript levels. b , c mRNA levels are arbitrarily expressed as FPKM (fragments per kilobase million). d Positive correlation between GABPA and FoxA1 protein expression in BC tumors. One hundred and twenty-four tumors from 112 patients with BC were analyzed for GABPA and FoxA1 protein levels using immunohistochemistry (IHC). Representative staining images (double positive and negative signals) were shown (Left and middle panels). Scale bars: 100 μm. The bottom panels: the enlarged areas of insets in the top panels. Right panel: the significant positive correlation between GABPA and FoxA1 expression as determined using IHC. e GABPA and FoxA1 expression as determined using IHC in adult human bladder. Shown are representatives of adjacent normal bladder tissues derived from patients with bladder cancer. GABPA signals are predominately localized in the bladder epithelium. Very similar expression profile of FoxA1 is shown here, too. f GABPA expression is associated with BC survival. Tumors derived from 45 BC patients were analyzed for their GABPA and FoxA1 expression using IHC and the medium level was used as a cutoff to separate low- and high-expression groups. GABPA and FoxA1 expression with survival association was determined by using univariate and multivariable analyses. OS overall survival
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    90
    Millipore rabbit anti gabpa
    AP-MS/MS identifies allele-specific protein-DNA interactions for hotspot <t>SDHD</t> promoter mutations. Custom oligos were 5’-biotinylated and designed to cover all analyzed mutation sites in the SDHD ). Each mutation site was analyzed by two independent label-swapped experiments. ELF1, <t>GABPA,</t> and GABPB1, if not observed as significant interactors, are colored in yellow in the background cloud and noted by name. A, AP-MS/MS analysis of the C523T SDHD promoter mutation interactors. Interactors with a ratio of at least 3 IQR (inter-quartile range) in both replicate experiments are colored in red. Ratios are shown after log2 transformation. Labels were swapped between replicates to avoid labeling bias, hence specific interactors show a high ratio in one experiment and a low ratio in the other. B, AP-MS/MS analysis of the C524T SDHD promoter mutation interactors. C, AP-MS/MS analysis of the C541T SDHD promoter mutation interactors. D, AP-MS/MS analysis of the C544T SDHD promoter mutation interactors. E–H, band-shift experiments confirm SDHD WT-specific GABP binding at C523T and C524T mutation sites, but not C541T or C544T. Oligos were shifted using recombinant human protein as described in the materials and methods. E, band-shift analysis with the C523T SDHD promoter mutation oligo and recombinant human GABP (GABPA and GABPB1) protein. Band-shift experiments for each protein-oligo combination were resolved on the same gel at the same exposure. The grey line indicates where a single lane was cropped out for clarity. F–H, band-shift analysis with (F) C524T, (G) C541T, and (H) C544T SDHD promoter mutation oligonucleotides and recombinant human GABP protein.
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    Abcam antibodies against gabpa
    AP-MS/MS identifies allele-specific protein-DNA interactions for hotspot <t>SDHD</t> promoter mutations. Custom oligos were 5’-biotinylated and designed to cover all analyzed mutation sites in the SDHD ). Each mutation site was analyzed by two independent label-swapped experiments. ELF1, <t>GABPA,</t> and GABPB1, if not observed as significant interactors, are colored in yellow in the background cloud and noted by name. A, AP-MS/MS analysis of the C523T SDHD promoter mutation interactors. Interactors with a ratio of at least 3 IQR (inter-quartile range) in both replicate experiments are colored in red. Ratios are shown after log2 transformation. Labels were swapped between replicates to avoid labeling bias, hence specific interactors show a high ratio in one experiment and a low ratio in the other. B, AP-MS/MS analysis of the C524T SDHD promoter mutation interactors. C, AP-MS/MS analysis of the C541T SDHD promoter mutation interactors. D, AP-MS/MS analysis of the C544T SDHD promoter mutation interactors. E–H, band-shift experiments confirm SDHD WT-specific GABP binding at C523T and C524T mutation sites, but not C541T or C544T. Oligos were shifted using recombinant human protein as described in the materials and methods. E, band-shift analysis with the C523T SDHD promoter mutation oligo and recombinant human GABP (GABPA and GABPB1) protein. Band-shift experiments for each protein-oligo combination were resolved on the same gel at the same exposure. The grey line indicates where a single lane was cropped out for clarity. F–H, band-shift analysis with (F) C524T, (G) C541T, and (H) C544T SDHD promoter mutation oligonucleotides and recombinant human GABP protein.
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    Image Search Results


    AMPK regulates HIF1α and GABPA through CREB1 a , ChIP using CREB1 antibody and quantitative PCR showing CREB1 binding to promoter/enhancer regions of HIF1α and GABPA. (n = 3). *p = 0.009; + 0.005. b , Quantification of HIF1α promoter activity in control (NT) and CREB1-silenced GSCs expressing HRE-firefly and renilla luciferase reporter plasmids. (n = 3). * p = 0.01. + 0.008. c , Q-RTPCR analysis of GSCs expressing NT or CREB1 shRNA. (n = 3). *p = 0.002; + 0.001; # 0.004; $ 0.007; **0.01; ++ 0.0005. d , Viability of GSCs expressing NT or CREB1 shRNA. (n = 3). *p = 0.003; + 0.01. Inset: WB of CREB1. e, f , ECAR and OCR in GSCs expressing CREB1 shRNA. (n = 3). *p = 0.01; + 0.006. g, h , WB of pCREB1 S133 and CREB1 (loading control) in GSCs expressing NTshRNA or two independent AMPKβ1shRNA. i , WB of pACC, pCREB1 and CREB1 in normal human astrocytes (NHA) treated with AMPK activator A769662 or glucose starvation (30 min). j , IHC of pCREB S133 in tumors derived from GSCs expressing NT or AMPKβ1 shRNA. Scale bar 100µm. k , Q-RTPCR analysis of GSC 326 expressing control virus, CREB1 S133A , or CREB1 S133E lentivirus. (n = 3). *p = 0.01; + ≤ 0.0003; $ 0.002; # 0.02; **0.03. I , WB of HIF1α and GABPA in GSCs expressing CREB1 shRNA, CREB1 S133A and CREB1 S133E mutants. Total CREB1 and actin are also shown. m , WB of HIF1α and GABPA in tumors derived from GSCs expressing S133A .

    Journal: Nature cell biology

    Article Title: AMP Kinase Promotes Glioblastoma Bioenergetics and Tumor Growth

    doi: 10.1038/s41556-018-0126-z

    Figure Lengend Snippet: AMPK regulates HIF1α and GABPA through CREB1 a , ChIP using CREB1 antibody and quantitative PCR showing CREB1 binding to promoter/enhancer regions of HIF1α and GABPA. (n = 3). *p = 0.009; + 0.005. b , Quantification of HIF1α promoter activity in control (NT) and CREB1-silenced GSCs expressing HRE-firefly and renilla luciferase reporter plasmids. (n = 3). * p = 0.01. + 0.008. c , Q-RTPCR analysis of GSCs expressing NT or CREB1 shRNA. (n = 3). *p = 0.002; + 0.001; # 0.004; $ 0.007; **0.01; ++ 0.0005. d , Viability of GSCs expressing NT or CREB1 shRNA. (n = 3). *p = 0.003; + 0.01. Inset: WB of CREB1. e, f , ECAR and OCR in GSCs expressing CREB1 shRNA. (n = 3). *p = 0.01; + 0.006. g, h , WB of pCREB1 S133 and CREB1 (loading control) in GSCs expressing NTshRNA or two independent AMPKβ1shRNA. i , WB of pACC, pCREB1 and CREB1 in normal human astrocytes (NHA) treated with AMPK activator A769662 or glucose starvation (30 min). j , IHC of pCREB S133 in tumors derived from GSCs expressing NT or AMPKβ1 shRNA. Scale bar 100µm. k , Q-RTPCR analysis of GSC 326 expressing control virus, CREB1 S133A , or CREB1 S133E lentivirus. (n = 3). *p = 0.01; + ≤ 0.0003; $ 0.002; # 0.02; **0.03. I , WB of HIF1α and GABPA in GSCs expressing CREB1 shRNA, CREB1 S133A and CREB1 S133E mutants. Total CREB1 and actin are also shown. m , WB of HIF1α and GABPA in tumors derived from GSCs expressing S133A .

    Article Snippet: Chromatin was sonicated to fragments of ~500 bp and immunoprecipitated using 1 microgram of ChIP-grade antibodies: HIF1α (NB100–134, Novus Biological), GABPA (sc-22810X, Santa Cruz Biotechnology ), CREB1 (9104, Cell Signaling Technology) and with irrelevant IgG antibodies of mouse and rabbit origin and recovered using protein A/G magnetic beads (from Magna ChIP™ A/G, Millipore).

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Activity Assay, Expressing, Luciferase, Reverse Transcription Polymerase Chain Reaction, shRNA, Western Blot, Immunohistochemistry, Derivative Assay

    GABPA expression is higher in the luminal subtype and associated with luminal signature, and predicts patient survival in bladder cancer (BC). a Schematic diagram showing luminal and basal BCs and their gene expression signatures. b Differential GABPA expression between luminal and basal subtypes of BCs. The TCGA cluster I (luminal) and III (basal) were compared for their GABPA and TERT (Telomerase reverse transcriptase) mRNA expression. Their mRNA levels are expressed as transcript reads determined using RNA sequencing. TERT telomerase reverse transcriptase. c Positive correlation between GABPA and FoxA1/GATA3 mRNA expression in BCs. The TCGA and GSE32894 cohorts of BC patients were analyzed for the relationship of GABPA with FoxA1 and GATA3 transcript levels. b , c mRNA levels are arbitrarily expressed as FPKM (fragments per kilobase million). d Positive correlation between GABPA and FoxA1 protein expression in BC tumors. One hundred and twenty-four tumors from 112 patients with BC were analyzed for GABPA and FoxA1 protein levels using immunohistochemistry (IHC). Representative staining images (double positive and negative signals) were shown (Left and middle panels). Scale bars: 100 μm. The bottom panels: the enlarged areas of insets in the top panels. Right panel: the significant positive correlation between GABPA and FoxA1 expression as determined using IHC. e GABPA and FoxA1 expression as determined using IHC in adult human bladder. Shown are representatives of adjacent normal bladder tissues derived from patients with bladder cancer. GABPA signals are predominately localized in the bladder epithelium. Very similar expression profile of FoxA1 is shown here, too. f GABPA expression is associated with BC survival. Tumors derived from 45 BC patients were analyzed for their GABPA and FoxA1 expression using IHC and the medium level was used as a cutoff to separate low- and high-expression groups. GABPA and FoxA1 expression with survival association was determined by using univariate and multivariable analyses. OS overall survival

    Journal: Cell Death and Differentiation

    Article Title: GABPA is a master regulator of luminal identity and restrains aggressive diseases in bladder cancer

    doi: 10.1038/s41418-019-0466-7

    Figure Lengend Snippet: GABPA expression is higher in the luminal subtype and associated with luminal signature, and predicts patient survival in bladder cancer (BC). a Schematic diagram showing luminal and basal BCs and their gene expression signatures. b Differential GABPA expression between luminal and basal subtypes of BCs. The TCGA cluster I (luminal) and III (basal) were compared for their GABPA and TERT (Telomerase reverse transcriptase) mRNA expression. Their mRNA levels are expressed as transcript reads determined using RNA sequencing. TERT telomerase reverse transcriptase. c Positive correlation between GABPA and FoxA1/GATA3 mRNA expression in BCs. The TCGA and GSE32894 cohorts of BC patients were analyzed for the relationship of GABPA with FoxA1 and GATA3 transcript levels. b , c mRNA levels are arbitrarily expressed as FPKM (fragments per kilobase million). d Positive correlation between GABPA and FoxA1 protein expression in BC tumors. One hundred and twenty-four tumors from 112 patients with BC were analyzed for GABPA and FoxA1 protein levels using immunohistochemistry (IHC). Representative staining images (double positive and negative signals) were shown (Left and middle panels). Scale bars: 100 μm. The bottom panels: the enlarged areas of insets in the top panels. Right panel: the significant positive correlation between GABPA and FoxA1 expression as determined using IHC. e GABPA and FoxA1 expression as determined using IHC in adult human bladder. Shown are representatives of adjacent normal bladder tissues derived from patients with bladder cancer. GABPA signals are predominately localized in the bladder epithelium. Very similar expression profile of FoxA1 is shown here, too. f GABPA expression is associated with BC survival. Tumors derived from 45 BC patients were analyzed for their GABPA and FoxA1 expression using IHC and the medium level was used as a cutoff to separate low- and high-expression groups. GABPA and FoxA1 expression with survival association was determined by using univariate and multivariable analyses. OS overall survival

    Article Snippet: Primary antibodies used for cell lines included anti-GABPA (1:1000 dilution, Santa Cruz, sc-22810), anti-FoxA1 (1:1000 dilution, Santa Cruz Biotechnology, Sc-514695), GATA3 (1:200 dilution, Abcam, ab14601), CDKN1A and 1B (1:1000 dilution, Santa Cruz Biotechnology), anti-OCT4 (1:500 dilution, Proteintech Group, Rosemont, IL, 11263-1-AP), anti-β-Actin (1:20,000 dilution, Santa Cruz Biotechnology, sc-47778), and anti-GAPDH (1:20,000 dilution, sc-47724 Santa Cruz Biotechnology).

    Techniques: Expressing, RNA Sequencing Assay, Immunohistochemistry, Staining, Derivative Assay

    GABPA regulates FoxA1 and GATA3 expression at transcriptional levels. Three independent experiments were performed. Bars: SD. a Downregulation of FoxA1 and GATA3 expression and their targets in BC cells depleted of GABPA. J82 and SW1710 cells were treated with two different GABPA siRNAs for 48 h and then analyzed for target gene expression at mRNA and protein levels. b Enhanced expression of FoxA1 and GATA3 in BC cells with GABPA overexpression. Ectopic GABPA was introduced into J82 and HT1197 cells and the target gene expression was then determined. c GABPA regulation of FoxA1 and GATA3 promoter activity. The upper panel: schematic presentation of FoxA1 and GATA3 promoters and locations of putative GABPA-binding motifs. The mutation of putative GABPA-binding motifs is marked in red. The bottom panel: GABPA depletion leads to the decline in wt but not mt promoter activity, while its overexpression promotes wt but not mt promoter activity. d The GABPA occupancy on FoxA1 and GATA3 promoter regions. Chromatin immunoprecipitation (ChIP) was performed to determine the presence of GABPA on the FoxA1 and GATA3 promoters. The TERT promoter with C228T was used as a positive control. Left: schematics of the GABPA-binding sites and primer locations in the promoter regions of TERT , FoxA1 , and GATA3 genes. Right: qPCR assessment of GABPA bound-promoter sequences in J82 cells treated with control or GABPA siRNA. C Control siRNA or vector, G1 and G GABPA siRNA and expression vector, respectively

    Journal: Cell Death and Differentiation

    Article Title: GABPA is a master regulator of luminal identity and restrains aggressive diseases in bladder cancer

    doi: 10.1038/s41418-019-0466-7

    Figure Lengend Snippet: GABPA regulates FoxA1 and GATA3 expression at transcriptional levels. Three independent experiments were performed. Bars: SD. a Downregulation of FoxA1 and GATA3 expression and their targets in BC cells depleted of GABPA. J82 and SW1710 cells were treated with two different GABPA siRNAs for 48 h and then analyzed for target gene expression at mRNA and protein levels. b Enhanced expression of FoxA1 and GATA3 in BC cells with GABPA overexpression. Ectopic GABPA was introduced into J82 and HT1197 cells and the target gene expression was then determined. c GABPA regulation of FoxA1 and GATA3 promoter activity. The upper panel: schematic presentation of FoxA1 and GATA3 promoters and locations of putative GABPA-binding motifs. The mutation of putative GABPA-binding motifs is marked in red. The bottom panel: GABPA depletion leads to the decline in wt but not mt promoter activity, while its overexpression promotes wt but not mt promoter activity. d The GABPA occupancy on FoxA1 and GATA3 promoter regions. Chromatin immunoprecipitation (ChIP) was performed to determine the presence of GABPA on the FoxA1 and GATA3 promoters. The TERT promoter with C228T was used as a positive control. Left: schematics of the GABPA-binding sites and primer locations in the promoter regions of TERT , FoxA1 , and GATA3 genes. Right: qPCR assessment of GABPA bound-promoter sequences in J82 cells treated with control or GABPA siRNA. C Control siRNA or vector, G1 and G GABPA siRNA and expression vector, respectively

    Article Snippet: Primary antibodies used for cell lines included anti-GABPA (1:1000 dilution, Santa Cruz, sc-22810), anti-FoxA1 (1:1000 dilution, Santa Cruz Biotechnology, Sc-514695), GATA3 (1:200 dilution, Abcam, ab14601), CDKN1A and 1B (1:1000 dilution, Santa Cruz Biotechnology), anti-OCT4 (1:500 dilution, Proteintech Group, Rosemont, IL, 11263-1-AP), anti-β-Actin (1:20,000 dilution, Santa Cruz Biotechnology, sc-47778), and anti-GAPDH (1:20,000 dilution, sc-47724 Santa Cruz Biotechnology).

    Techniques: Expressing, Over Expression, Activity Assay, Binding Assay, Mutagenesis, Chromatin Immunoprecipitation, Positive Control, Real-time Polymerase Chain Reaction, Plasmid Preparation

    GABPA regulates invasion and stem cell phenotypes, and cisplatin sensitivity of BC cells. Three independent experiments were performed. Bars: SD. a , b GABPA depletion-mediated robust increases in BC cell invasion. J82 (left) and SW1710 cells (right) were treated with GABPA siRNA for 48 h and their invasive capability was evaluated using the matrigel assay. Cells passing matrigel were stained, counted, and photographed. c , d J82 and HT1197 cells were transfected with GABPA expression vectors and these cells were then analyzed as above. e , f , g GABPA regulation of BC stem cell self-renewal. Left panel: GABPA was knocked down in J82 cells using its specific siRNAs and sphere formation was then assessed. Middle and right panels: J82 and HT1197 cells with ectopic GABPA expression were assessed for their sphere formation. h FoxA1 effect on invasive and stem cell phenotypes of BC cells. J82 cells were depleted of FoxA1 expression using its specific siRNA and assessed for their abilities of invasion and self-renewal. i The abolishment of GABPA-mediated inhibition of J82 cell invasion via FoxA1 depletion. Control and GABPA overexpressed J82 cells were treated with FoxA1 siRNA and invasion then evaluated. j GABPA depletion-mediated cisplatin resistance to BC cells. J82 and SW1710 cells were treated with two different GABPA siRNAs and then incubated with various concentrations of cisplatin. Cell viability was determined using the MTT assay. Scale bars: 20 μm. C: Control; G1 and G2, GABPA siRNA1 and 2; G: GABPA expression vector

    Journal: Cell Death and Differentiation

    Article Title: GABPA is a master regulator of luminal identity and restrains aggressive diseases in bladder cancer

    doi: 10.1038/s41418-019-0466-7

    Figure Lengend Snippet: GABPA regulates invasion and stem cell phenotypes, and cisplatin sensitivity of BC cells. Three independent experiments were performed. Bars: SD. a , b GABPA depletion-mediated robust increases in BC cell invasion. J82 (left) and SW1710 cells (right) were treated with GABPA siRNA for 48 h and their invasive capability was evaluated using the matrigel assay. Cells passing matrigel were stained, counted, and photographed. c , d J82 and HT1197 cells were transfected with GABPA expression vectors and these cells were then analyzed as above. e , f , g GABPA regulation of BC stem cell self-renewal. Left panel: GABPA was knocked down in J82 cells using its specific siRNAs and sphere formation was then assessed. Middle and right panels: J82 and HT1197 cells with ectopic GABPA expression were assessed for their sphere formation. h FoxA1 effect on invasive and stem cell phenotypes of BC cells. J82 cells were depleted of FoxA1 expression using its specific siRNA and assessed for their abilities of invasion and self-renewal. i The abolishment of GABPA-mediated inhibition of J82 cell invasion via FoxA1 depletion. Control and GABPA overexpressed J82 cells were treated with FoxA1 siRNA and invasion then evaluated. j GABPA depletion-mediated cisplatin resistance to BC cells. J82 and SW1710 cells were treated with two different GABPA siRNAs and then incubated with various concentrations of cisplatin. Cell viability was determined using the MTT assay. Scale bars: 20 μm. C: Control; G1 and G2, GABPA siRNA1 and 2; G: GABPA expression vector

    Article Snippet: Primary antibodies used for cell lines included anti-GABPA (1:1000 dilution, Santa Cruz, sc-22810), anti-FoxA1 (1:1000 dilution, Santa Cruz Biotechnology, Sc-514695), GATA3 (1:200 dilution, Abcam, ab14601), CDKN1A and 1B (1:1000 dilution, Santa Cruz Biotechnology), anti-OCT4 (1:500 dilution, Proteintech Group, Rosemont, IL, 11263-1-AP), anti-β-Actin (1:20,000 dilution, Santa Cruz Biotechnology, sc-47778), and anti-GAPDH (1:20,000 dilution, sc-47724 Santa Cruz Biotechnology).

    Techniques: Matrigel Assay, Staining, Transfection, Expressing, Inhibition, Incubation, MTT Assay, Plasmid Preparation

    AP-MS/MS identifies allele-specific protein-DNA interactions for hotspot SDHD promoter mutations. Custom oligos were 5’-biotinylated and designed to cover all analyzed mutation sites in the SDHD ). Each mutation site was analyzed by two independent label-swapped experiments. ELF1, GABPA, and GABPB1, if not observed as significant interactors, are colored in yellow in the background cloud and noted by name. A, AP-MS/MS analysis of the C523T SDHD promoter mutation interactors. Interactors with a ratio of at least 3 IQR (inter-quartile range) in both replicate experiments are colored in red. Ratios are shown after log2 transformation. Labels were swapped between replicates to avoid labeling bias, hence specific interactors show a high ratio in one experiment and a low ratio in the other. B, AP-MS/MS analysis of the C524T SDHD promoter mutation interactors. C, AP-MS/MS analysis of the C541T SDHD promoter mutation interactors. D, AP-MS/MS analysis of the C544T SDHD promoter mutation interactors. E–H, band-shift experiments confirm SDHD WT-specific GABP binding at C523T and C524T mutation sites, but not C541T or C544T. Oligos were shifted using recombinant human protein as described in the materials and methods. E, band-shift analysis with the C523T SDHD promoter mutation oligo and recombinant human GABP (GABPA and GABPB1) protein. Band-shift experiments for each protein-oligo combination were resolved on the same gel at the same exposure. The grey line indicates where a single lane was cropped out for clarity. F–H, band-shift analysis with (F) C524T, (G) C541T, and (H) C544T SDHD promoter mutation oligonucleotides and recombinant human GABP protein.

    Journal: Cancer research

    Article Title: SDHD promoter mutations ablate GABP transcription factor binding in melanoma

    doi: 10.1158/0008-5472.CAN-16-0919

    Figure Lengend Snippet: AP-MS/MS identifies allele-specific protein-DNA interactions for hotspot SDHD promoter mutations. Custom oligos were 5’-biotinylated and designed to cover all analyzed mutation sites in the SDHD ). Each mutation site was analyzed by two independent label-swapped experiments. ELF1, GABPA, and GABPB1, if not observed as significant interactors, are colored in yellow in the background cloud and noted by name. A, AP-MS/MS analysis of the C523T SDHD promoter mutation interactors. Interactors with a ratio of at least 3 IQR (inter-quartile range) in both replicate experiments are colored in red. Ratios are shown after log2 transformation. Labels were swapped between replicates to avoid labeling bias, hence specific interactors show a high ratio in one experiment and a low ratio in the other. B, AP-MS/MS analysis of the C524T SDHD promoter mutation interactors. C, AP-MS/MS analysis of the C541T SDHD promoter mutation interactors. D, AP-MS/MS analysis of the C544T SDHD promoter mutation interactors. E–H, band-shift experiments confirm SDHD WT-specific GABP binding at C523T and C524T mutation sites, but not C541T or C544T. Oligos were shifted using recombinant human protein as described in the materials and methods. E, band-shift analysis with the C523T SDHD promoter mutation oligo and recombinant human GABP (GABPA and GABPB1) protein. Band-shift experiments for each protein-oligo combination were resolved on the same gel at the same exposure. The grey line indicates where a single lane was cropped out for clarity. F–H, band-shift analysis with (F) C524T, (G) C541T, and (H) C544T SDHD promoter mutation oligonucleotides and recombinant human GABP protein.

    Article Snippet: The primary antibodies used were rabbit anti-SDHD (ab189945, Abcam), rabbit anti-GABPA (ABE1047, Millipore), mouse anti-GABPB1 (sc271571, Santa Cruz Biotechnology), and mouse anti-β-actin (A5316, Sigma-Aldrich).

    Techniques: Tandem Mass Spectroscopy, Mutagenesis, Transformation Assay, Labeling, Electrophoretic Mobility Shift Assay, Binding Assay, Recombinant

    Effect of siRNA-mediated knockdown of GABPA and GABPB1 on SDHD protein levels. A, concomitant depletion of both GABPA and GABPB1 or GABPA alone decreases SDHD expression at the protein level in both UACC903 and UACC1113. B, depletion of GABPB1 did not change SDHD expression at the protein level in both UACC903 and UACC1113, nor does it dramatically increase GABPA protein levels. D2, D5 and D7 denote the number of days after initial transfection in knockdown experiments.

    Journal: Cancer research

    Article Title: SDHD promoter mutations ablate GABP transcription factor binding in melanoma

    doi: 10.1158/0008-5472.CAN-16-0919

    Figure Lengend Snippet: Effect of siRNA-mediated knockdown of GABPA and GABPB1 on SDHD protein levels. A, concomitant depletion of both GABPA and GABPB1 or GABPA alone decreases SDHD expression at the protein level in both UACC903 and UACC1113. B, depletion of GABPB1 did not change SDHD expression at the protein level in both UACC903 and UACC1113, nor does it dramatically increase GABPA protein levels. D2, D5 and D7 denote the number of days after initial transfection in knockdown experiments.

    Article Snippet: The primary antibodies used were rabbit anti-SDHD (ab189945, Abcam), rabbit anti-GABPA (ABE1047, Millipore), mouse anti-GABPB1 (sc271571, Santa Cruz Biotechnology), and mouse anti-β-actin (A5316, Sigma-Aldrich).

    Techniques: Expressing, Transfection

    siRNA-mediated knockdown of GABPA and GABPB1 deregulates SDHD expression in melanoma cells. A, GABPA depletion decreases SDHD and increases GABPB1 expression in three melanoma cell lines (UACC1113, UACC903, and C021). B, GABPB1 depletion increases GABPA expression in three melanoma cell lines (UACC1113, UACC903 and C021), but has little effect on SDHD levels. C, concomitant depletion of both GABPA and GABPB1 decreases SDHD expression in both UACC1113 and UACC903 cell lines.

    Journal: Cancer research

    Article Title: SDHD promoter mutations ablate GABP transcription factor binding in melanoma

    doi: 10.1158/0008-5472.CAN-16-0919

    Figure Lengend Snippet: siRNA-mediated knockdown of GABPA and GABPB1 deregulates SDHD expression in melanoma cells. A, GABPA depletion decreases SDHD and increases GABPB1 expression in three melanoma cell lines (UACC1113, UACC903, and C021). B, GABPB1 depletion increases GABPA expression in three melanoma cell lines (UACC1113, UACC903 and C021), but has little effect on SDHD levels. C, concomitant depletion of both GABPA and GABPB1 decreases SDHD expression in both UACC1113 and UACC903 cell lines.

    Article Snippet: The primary antibodies used were rabbit anti-SDHD (ab189945, Abcam), rabbit anti-GABPA (ABE1047, Millipore), mouse anti-GABPB1 (sc271571, Santa Cruz Biotechnology), and mouse anti-β-actin (A5316, Sigma-Aldrich).

    Techniques: Expressing