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    Name:
    SP1 D4C3 Rabbit mAb
    Description:
    Specificity protein 1 SP1 is a ubiquitously expressed transcription factor belonging to the family of C2H2 type zinc finger containing DNA binding proteins SP1 binds GC rich motifs with high affinity and regulates the expression of numerous mammalian genes 1 2 It interacts with many other transcription factors such as c Myc EGR1 and Stat1 and with basal transcription machinery components SP1 interacts with chromatin modifying factors such as histone deacetylases HDACs and p300 in chromatin remodeling Transcriptional activity and stability of SP1 are regulated by post translational modification including phosphorylation acetylation ubiquitination and glycosylation 3 Glycosylation of SP1 following insulin treatment leads to increased nuclear localization while glucagon treatment increases cytoplasmic SP1 levels 4 6 Investigators have found high levels of SP1 in patients with Alzheimer s disease 7
    Catalog Number:
    9389
    Price:
    None
    Applications:
    Western Blot, Immunoprecipitation, Immunohistochemistry, Flow Cytometry, Chromatin Immunoprecipitation
    Category:
    Primary Antibodies
    Source:
    Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro593 of human SP1 protein (Isoform 1).
    Reactivity:
    Human Monkey
    Buy from Supplier


    Structured Review

    Cell Signaling Technology Inc sp1 d4c3
    Specificity protein 1 SP1 is a ubiquitously expressed transcription factor belonging to the family of C2H2 type zinc finger containing DNA binding proteins SP1 binds GC rich motifs with high affinity and regulates the expression of numerous mammalian genes 1 2 It interacts with many other transcription factors such as c Myc EGR1 and Stat1 and with basal transcription machinery components SP1 interacts with chromatin modifying factors such as histone deacetylases HDACs and p300 in chromatin remodeling Transcriptional activity and stability of SP1 are regulated by post translational modification including phosphorylation acetylation ubiquitination and glycosylation 3 Glycosylation of SP1 following insulin treatment leads to increased nuclear localization while glucagon treatment increases cytoplasmic SP1 levels 4 6 Investigators have found high levels of SP1 in patients with Alzheimer s disease 7
    https://www.bioz.com/result/sp1 d4c3/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sp1 d4c3 - by Bioz Stars, 2020-08
    94/100 stars

    Images

    Related Articles

    Western Blot:

    Article Title: Inhibition of the hexosamine biosynthetic pathway promotes castration-resistant prostate cancer
    Article Snippet: .. Western blot Antibodies to GNPNAT1 (Proteintech, cat # 16282-1-AP, 1:2,000), GFPT1 (Santa Cruz Biotechnology, cat# sc-377479), SP1 (Cell Signaling, cat # 9389, 1:1000), ChREBP (Novus, cat # NB400-136V, 1:1,000), AR (Santa Cruz Biotechnology, cat # sc-816, sc-7305), AR-V7 (Precision Antibody, cat # AG10008), PI3K-p85 (BD Biosciences, Cat # 610045) and Actin (used as loading control; Sigma, 1:7500) were used. ..

    Incubation:

    Article Title: Elevated histone H3 acetylation and loss of the Sp1–HDAC1 complex de-repress the GM2-synthase gene in renal cell carcinoma
    Article Snippet: .. The supernatants were incubated overnight with antibodies specific for Sp1 (D4C3, 9389) (Cell Signaling Technology). .. Normal rabbit IgG (3900S) (Cell Signaling Technology) was taken as an isotype control for immunoprecipitation.

    Article Title: Western blot data using two distinct anti-O-GlcNAc monoclonal antibodies showing unique glycosylation status on cellular proteins under 2-deoxy-d-glucose treatment
    Article Snippet: .. 2.3 Immunoprecipitation The whole cell lysates (0.2 ml) were incubated with 4 μl (1 μg) of rabbit anti-Sp1 monoclonal antibody (D4C3, Cell Signaling Technology, Danvers, MA) at 4 °C overnight, and subsequently incubated with 20 μl of 50% protein A sepharose bead slurry (GE Healthcare) at 4 °C for 3 h. The samples were centrifuged at 14,000 g for 1 min at 4 °C, and the precipitates (beads) were washed five times with 0.5 ml of wash buffer (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3 VO4 , 1 μg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 50 μM PUGNAc) and were finally resuspended in 20 μl of SDS-PAGE sample buffer. .. 2.4 Western blotting The whole cell lysates (5 μg of total protein) and anti-Sp1 antibody precipitates were separated by SDS-PAGE, and the proteins in the gel were transferred onto Immobilon-P PVDF membranes (Millipore, Billerica, MA) by electroblotting at a constant current of 90 mA for 1 h. After blotting, the membrane was incubated with primary antibody and subsequently incubated with a horseradish peroxidase-linked secondary antibody.

    other:

    Article Title: Activated MEK/ERK Pathway Drives Widespread and Coordinated Overexpression of UHRF1 and DNMT1 in Cancer cells
    Article Snippet: The antibodies used in this study were as follows: Erk1/2 (4695), P-ERK1/2 (4370), E2F1 (3742) and SP1 (9389) from Cell Signaling Technology (Danvers, MA); DNMT1 (sc-20701) and DNMT3A (sc-20703) from Santa Cruz Biotechnology (Dallas, TX); UHRF1 (21402-1-AP) from Proteintech (Rosemont, IL); β-actin (M1210-2) from HUABIO (Cambridge, MA) and GAPDH (3B3) from Abmart (Berkeley Heights, NJ).

    SDS Page:

    Article Title: Western blot data using two distinct anti-O-GlcNAc monoclonal antibodies showing unique glycosylation status on cellular proteins under 2-deoxy-d-glucose treatment
    Article Snippet: .. 2.3 Immunoprecipitation The whole cell lysates (0.2 ml) were incubated with 4 μl (1 μg) of rabbit anti-Sp1 monoclonal antibody (D4C3, Cell Signaling Technology, Danvers, MA) at 4 °C overnight, and subsequently incubated with 20 μl of 50% protein A sepharose bead slurry (GE Healthcare) at 4 °C for 3 h. The samples were centrifuged at 14,000 g for 1 min at 4 °C, and the precipitates (beads) were washed five times with 0.5 ml of wash buffer (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3 VO4 , 1 μg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 50 μM PUGNAc) and were finally resuspended in 20 μl of SDS-PAGE sample buffer. .. 2.4 Western blotting The whole cell lysates (5 μg of total protein) and anti-Sp1 antibody precipitates were separated by SDS-PAGE, and the proteins in the gel were transferred onto Immobilon-P PVDF membranes (Millipore, Billerica, MA) by electroblotting at a constant current of 90 mA for 1 h. After blotting, the membrane was incubated with primary antibody and subsequently incubated with a horseradish peroxidase-linked secondary antibody.

    Immunoprecipitation:

    Article Title: Western blot data using two distinct anti-O-GlcNAc monoclonal antibodies showing unique glycosylation status on cellular proteins under 2-deoxy-d-glucose treatment
    Article Snippet: .. 2.3 Immunoprecipitation The whole cell lysates (0.2 ml) were incubated with 4 μl (1 μg) of rabbit anti-Sp1 monoclonal antibody (D4C3, Cell Signaling Technology, Danvers, MA) at 4 °C overnight, and subsequently incubated with 20 μl of 50% protein A sepharose bead slurry (GE Healthcare) at 4 °C for 3 h. The samples were centrifuged at 14,000 g for 1 min at 4 °C, and the precipitates (beads) were washed five times with 0.5 ml of wash buffer (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3 VO4 , 1 μg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 50 μM PUGNAc) and were finally resuspended in 20 μl of SDS-PAGE sample buffer. .. 2.4 Western blotting The whole cell lysates (5 μg of total protein) and anti-Sp1 antibody precipitates were separated by SDS-PAGE, and the proteins in the gel were transferred onto Immobilon-P PVDF membranes (Millipore, Billerica, MA) by electroblotting at a constant current of 90 mA for 1 h. After blotting, the membrane was incubated with primary antibody and subsequently incubated with a horseradish peroxidase-linked secondary antibody.

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  • 90
    Cell Signaling Technology Inc chromatin immunoprecipitation chip qpcr sp1 chip qpcr
    Chromatin structure and DNA-protein interactions surrounding the 9p22 PFS-associated SNPs A. Colored histograms denote histone modification ChIP-seq data from UCSD and ENCODE. Epigenetic marks for H3K4me1 and H3K27ac in ovary from UCSD and 7 cell types from ENCODE, and transcription factor ChIP-seq data from ENCODE are shown. The grey shaded region denotes the PRE containing SNPs rs72700653 and rs7874043. B. EMSA for oligonucleotides containing SNP rs7874043 with the A = common allele and C = minor allele as indicated below the panel, assayed using JAM and A2780 nuclear extracts. Labels above each lane indicate inclusion of competitor oligonucleotides at 30-fold molar excess: (−) no competitor (Lanes 1,2,8,9); Self-C allele (Lanes 3,10), AP1 (Lanes 4,11), FOXA1 (Lanes 5,12), <t>Sp1</t> (Lanes 6,13) and a control sequence (Lanes 7,14; containing binding site for ATF, a TF not predicted to bind). The Sp1-containing complexes are indicated with red arrowheads. C. <t>ChIP-qPCR</t> on the PRE in JAM and A2780 cell lines. ChIP assays were performed with Sp1 antibodies or non-immune IgG, with a region 2.3kb upstream of the predicted Sp1-binding site (Control) used as a control for nonspecific binding. Graphs represent two biological replicates. Error bars denote SD.
    Chromatin Immunoprecipitation Chip Qpcr Sp1 Chip Qpcr, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/chromatin immunoprecipitation chip qpcr sp1 chip qpcr/product/Cell Signaling Technology Inc
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    chromatin immunoprecipitation chip qpcr sp1 chip qpcr - by Bioz Stars, 2020-08
    90/100 stars
      Buy from Supplier

    94
    Cell Signaling Technology Inc sp1
    Multiple downstream transcription factors contribute to transcriptional activation of UHRF1 and DNMT1 by the MEK/ERK pathway. ( a , b ) The diagrams illustrating the putative binding sites for transcription factors AML1a, E2F1, <t>SP1,</t> STAT3 and YY1 within the region of −5kb and + 3 kb around the transcriptional start site of UHRF1 gene ( a ) and DNMT1 gene ( b ). ( c ) qRT-PCR analysis showing efficient knockdown of corresponding transcription factors in HCT116 cells by specific siRNA against AML1a, E2F1, SP1, STAT3 and YY1, respectively. ( d ) qRT-PCR analysis showing the effect on UHRF1 and DNMT1 expression in HCT116 cells upon knockdown of each of the transcription factors. ( e , f ) ChIP analysis showing the effect of PD0325901 treatment on the binding of E2F1 and SP1 to two regions within the UHRF1 promoter ( e ) or DNMT1 promoter ( f ). IgG, control immunoglobulin. ( g , h ). Luciferase reporter assay showing that E2F1 and SP1 markedly enhanced transcriptional activation from the UHRF1 and DNMT1 promoters. The control PGL3-luc and UHRF1-luc ( g ) or DNMT1-luc ( h ) were cotransfected with or without an E2F1 or SP1 expression plasmid into HCT116 cells and treated with or without PD0325901 as indicated. Also shown were western blotting data for corresponding samples.
    Sp1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sp1/product/Cell Signaling Technology Inc
    Average 94 stars, based on 19 article reviews
    Price from $9.99 to $1999.99
    sp1 - by Bioz Stars, 2020-08
    94/100 stars
      Buy from Supplier

    Image Search Results


    Chromatin structure and DNA-protein interactions surrounding the 9p22 PFS-associated SNPs A. Colored histograms denote histone modification ChIP-seq data from UCSD and ENCODE. Epigenetic marks for H3K4me1 and H3K27ac in ovary from UCSD and 7 cell types from ENCODE, and transcription factor ChIP-seq data from ENCODE are shown. The grey shaded region denotes the PRE containing SNPs rs72700653 and rs7874043. B. EMSA for oligonucleotides containing SNP rs7874043 with the A = common allele and C = minor allele as indicated below the panel, assayed using JAM and A2780 nuclear extracts. Labels above each lane indicate inclusion of competitor oligonucleotides at 30-fold molar excess: (−) no competitor (Lanes 1,2,8,9); Self-C allele (Lanes 3,10), AP1 (Lanes 4,11), FOXA1 (Lanes 5,12), Sp1 (Lanes 6,13) and a control sequence (Lanes 7,14; containing binding site for ATF, a TF not predicted to bind). The Sp1-containing complexes are indicated with red arrowheads. C. ChIP-qPCR on the PRE in JAM and A2780 cell lines. ChIP assays were performed with Sp1 antibodies or non-immune IgG, with a region 2.3kb upstream of the predicted Sp1-binding site (Control) used as a control for nonspecific binding. Graphs represent two biological replicates. Error bars denote SD.

    Journal: Oncotarget

    Article Title: Germline polymorphisms in an enhancer of PSIP1 are associated with progression-free survival in epithelial ovarian cancer

    doi: 10.18632/oncotarget.7047

    Figure Lengend Snippet: Chromatin structure and DNA-protein interactions surrounding the 9p22 PFS-associated SNPs A. Colored histograms denote histone modification ChIP-seq data from UCSD and ENCODE. Epigenetic marks for H3K4me1 and H3K27ac in ovary from UCSD and 7 cell types from ENCODE, and transcription factor ChIP-seq data from ENCODE are shown. The grey shaded region denotes the PRE containing SNPs rs72700653 and rs7874043. B. EMSA for oligonucleotides containing SNP rs7874043 with the A = common allele and C = minor allele as indicated below the panel, assayed using JAM and A2780 nuclear extracts. Labels above each lane indicate inclusion of competitor oligonucleotides at 30-fold molar excess: (−) no competitor (Lanes 1,2,8,9); Self-C allele (Lanes 3,10), AP1 (Lanes 4,11), FOXA1 (Lanes 5,12), Sp1 (Lanes 6,13) and a control sequence (Lanes 7,14; containing binding site for ATF, a TF not predicted to bind). The Sp1-containing complexes are indicated with red arrowheads. C. ChIP-qPCR on the PRE in JAM and A2780 cell lines. ChIP assays were performed with Sp1 antibodies or non-immune IgG, with a region 2.3kb upstream of the predicted Sp1-binding site (Control) used as a control for nonspecific binding. Graphs represent two biological replicates. Error bars denote SD.

    Article Snippet: Chromatin immunoprecipitation (ChIP) qPCR Sp1 ChIP-qPCR (Sp1; D4C3 rabbit monoclonal, Cell Signalling) assays were conducted as described previously [ ] with a sheared fragment size of 300 bp to 1 kb.

    Techniques: Modification, Chromatin Immunoprecipitation, Sequencing, Binding Assay, Real-time Polymerase Chain Reaction

    Multiple downstream transcription factors contribute to transcriptional activation of UHRF1 and DNMT1 by the MEK/ERK pathway. ( a , b ) The diagrams illustrating the putative binding sites for transcription factors AML1a, E2F1, SP1, STAT3 and YY1 within the region of −5kb and + 3 kb around the transcriptional start site of UHRF1 gene ( a ) and DNMT1 gene ( b ). ( c ) qRT-PCR analysis showing efficient knockdown of corresponding transcription factors in HCT116 cells by specific siRNA against AML1a, E2F1, SP1, STAT3 and YY1, respectively. ( d ) qRT-PCR analysis showing the effect on UHRF1 and DNMT1 expression in HCT116 cells upon knockdown of each of the transcription factors. ( e , f ) ChIP analysis showing the effect of PD0325901 treatment on the binding of E2F1 and SP1 to two regions within the UHRF1 promoter ( e ) or DNMT1 promoter ( f ). IgG, control immunoglobulin. ( g , h ). Luciferase reporter assay showing that E2F1 and SP1 markedly enhanced transcriptional activation from the UHRF1 and DNMT1 promoters. The control PGL3-luc and UHRF1-luc ( g ) or DNMT1-luc ( h ) were cotransfected with or without an E2F1 or SP1 expression plasmid into HCT116 cells and treated with or without PD0325901 as indicated. Also shown were western blotting data for corresponding samples.

    Journal: Scientific Reports

    Article Title: Activated MEK/ERK Pathway Drives Widespread and Coordinated Overexpression of UHRF1 and DNMT1 in Cancer cells

    doi: 10.1038/s41598-018-37258-3

    Figure Lengend Snippet: Multiple downstream transcription factors contribute to transcriptional activation of UHRF1 and DNMT1 by the MEK/ERK pathway. ( a , b ) The diagrams illustrating the putative binding sites for transcription factors AML1a, E2F1, SP1, STAT3 and YY1 within the region of −5kb and + 3 kb around the transcriptional start site of UHRF1 gene ( a ) and DNMT1 gene ( b ). ( c ) qRT-PCR analysis showing efficient knockdown of corresponding transcription factors in HCT116 cells by specific siRNA against AML1a, E2F1, SP1, STAT3 and YY1, respectively. ( d ) qRT-PCR analysis showing the effect on UHRF1 and DNMT1 expression in HCT116 cells upon knockdown of each of the transcription factors. ( e , f ) ChIP analysis showing the effect of PD0325901 treatment on the binding of E2F1 and SP1 to two regions within the UHRF1 promoter ( e ) or DNMT1 promoter ( f ). IgG, control immunoglobulin. ( g , h ). Luciferase reporter assay showing that E2F1 and SP1 markedly enhanced transcriptional activation from the UHRF1 and DNMT1 promoters. The control PGL3-luc and UHRF1-luc ( g ) or DNMT1-luc ( h ) were cotransfected with or without an E2F1 or SP1 expression plasmid into HCT116 cells and treated with or without PD0325901 as indicated. Also shown were western blotting data for corresponding samples.

    Article Snippet: The antibodies used in this study were as follows: Erk1/2 (4695), P-ERK1/2 (4370), E2F1 (3742) and SP1 (9389) from Cell Signaling Technology (Danvers, MA); DNMT1 (sc-20701) and DNMT3A (sc-20703) from Santa Cruz Biotechnology (Dallas, TX); UHRF1 (21402-1-AP) from Proteintech (Rosemont, IL); β-actin (M1210-2) from HUABIO (Cambridge, MA) and GAPDH (3B3) from Abmart (Berkeley Heights, NJ).

    Techniques: Activation Assay, Binding Assay, Quantitative RT-PCR, Expressing, Chromatin Immunoprecipitation, Luciferase, Reporter Assay, Plasmid Preparation, Western Blot

    Multiple downstream transcription factors contribute to transcriptional activation of UHRF1 and DNMT1 by the MEK/ERK pathway. ( a , b ) The diagrams illustrating the putative binding sites for transcription factors AML1a, E2F1, SP1, STAT3 and YY1 within the region of −5kb and + 3 kb around the transcriptional start site of UHRF1 gene ( a ) and DNMT1 gene ( b ). ( c ) qRT-PCR analysis showing efficient knockdown of corresponding transcription factors in HCT116 cells by specific siRNA against AML1a, E2F1, SP1, STAT3 and YY1, respectively. ( d ) qRT-PCR analysis showing the effect on UHRF1 and DNMT1 expression in HCT116 cells upon knockdown of each of the transcription factors. ( e , f ) ChIP analysis showing the effect of PD0325901 treatment on the binding of E2F1 and SP1 to two regions within the UHRF1 promoter ( e ) or DNMT1 promoter ( f ). IgG, control immunoglobulin. ( g , h ). Luciferase reporter assay showing that E2F1 and SP1 markedly enhanced transcriptional activation from the UHRF1 and DNMT1 promoters. The control PGL3-luc and UHRF1-luc ( g ) or DNMT1-luc ( h ) were cotransfected with or without an E2F1 or SP1 expression plasmid into HCT116 cells and treated with or without PD0325901 as indicated. Also shown were western blotting data for corresponding samples.

    Journal: Scientific Reports

    Article Title: Activated MEK/ERK Pathway Drives Widespread and Coordinated Overexpression of UHRF1 and DNMT1 in Cancer cells

    doi: 10.1038/s41598-018-37258-3

    Figure Lengend Snippet: Multiple downstream transcription factors contribute to transcriptional activation of UHRF1 and DNMT1 by the MEK/ERK pathway. ( a , b ) The diagrams illustrating the putative binding sites for transcription factors AML1a, E2F1, SP1, STAT3 and YY1 within the region of −5kb and + 3 kb around the transcriptional start site of UHRF1 gene ( a ) and DNMT1 gene ( b ). ( c ) qRT-PCR analysis showing efficient knockdown of corresponding transcription factors in HCT116 cells by specific siRNA against AML1a, E2F1, SP1, STAT3 and YY1, respectively. ( d ) qRT-PCR analysis showing the effect on UHRF1 and DNMT1 expression in HCT116 cells upon knockdown of each of the transcription factors. ( e , f ) ChIP analysis showing the effect of PD0325901 treatment on the binding of E2F1 and SP1 to two regions within the UHRF1 promoter ( e ) or DNMT1 promoter ( f ). IgG, control immunoglobulin. ( g , h ). Luciferase reporter assay showing that E2F1 and SP1 markedly enhanced transcriptional activation from the UHRF1 and DNMT1 promoters. The control PGL3-luc and UHRF1-luc ( g ) or DNMT1-luc ( h ) were cotransfected with or without an E2F1 or SP1 expression plasmid into HCT116 cells and treated with or without PD0325901 as indicated. Also shown were western blotting data for corresponding samples.

    Article Snippet: The antibodies used in this study were as follows: Erk1/2 (4695), P-ERK1/2 (4370), E2F1 (3742) and SP1 (9389) from Cell Signaling Technology (Danvers, MA); DNMT1 (sc-20701) and DNMT3A (sc-20703) from Santa Cruz Biotechnology (Dallas, TX); UHRF1 (21402-1-AP) from Proteintech (Rosemont, IL); β-actin (M1210-2) from HUABIO (Cambridge, MA) and GAPDH (3B3) from Abmart (Berkeley Heights, NJ).

    Techniques: Activation Assay, Binding Assay, Quantitative RT-PCR, Expressing, Chromatin Immunoprecipitation, Luciferase, Reporter Assay, Plasmid Preparation, Western Blot

    Western blot analysis of Sp1 protein in 2DG-treated NCCIT cells. The whole cell lysates of NCCIT cells treated with 2DG for the indicated times (0, 24, 72, or 168 h) were precipitated with an anti-Sp1 antibody (D4C3), and the precipitated proteins were immunoblotted with D4C3, anti- O -GlcNAc antibodies (RL2 or CTD110.6), anti-Phosphoserine/threonine (P-Ser/Thr), anti-SUMO1, or anti-Ubiquitin. These transcriptional modifications were observed in Sp1 proteins [6] . Among these modifications, only O -GlcNAcylation was clearly detected in the D4C3 precipitates. The Sp1 levels in whole cell lysates were shown as a reference (left panel).

    Journal: Data in Brief

    Article Title: Western blot data using two distinct anti-O-GlcNAc monoclonal antibodies showing unique glycosylation status on cellular proteins under 2-deoxy-d-glucose treatment

    doi: 10.1016/j.dib.2016.12.001

    Figure Lengend Snippet: Western blot analysis of Sp1 protein in 2DG-treated NCCIT cells. The whole cell lysates of NCCIT cells treated with 2DG for the indicated times (0, 24, 72, or 168 h) were precipitated with an anti-Sp1 antibody (D4C3), and the precipitated proteins were immunoblotted with D4C3, anti- O -GlcNAc antibodies (RL2 or CTD110.6), anti-Phosphoserine/threonine (P-Ser/Thr), anti-SUMO1, or anti-Ubiquitin. These transcriptional modifications were observed in Sp1 proteins [6] . Among these modifications, only O -GlcNAcylation was clearly detected in the D4C3 precipitates. The Sp1 levels in whole cell lysates were shown as a reference (left panel).

    Article Snippet: The primary antibodies used were mouse anti-O -GlcNAc monoclonal antibodies (RL2, Thermo Fisher Scientific, Waltham, MA; CTD110.6, Cell Signaling Technology), rabbit anti-Sp1 monoclonal antibody (D4C3, Cell Signaling Technology), rabbit anti-GRP78/Bip monoclonal antibody (C50B12, Cell Signaling Technology), rabbit anti-GAPDH monoclonal antibody (D16H11, Cell Signaling Technology), rabbit anti-β-Actin polyclonal antibody (#4967, Cell Signaling Technology), rabbit anti-Phosphoserine/threonine polyclonal antibody (ab17464, abcam, Cambridge, UK), mouse anti-SUMO1 monoclonal antibody (21C7, BostonBiochem, Cambridge, MA), and mouse anti-Ubiquitin monoclonal antibody (P4D1, Cell Signaling Technology).

    Techniques: Western Blot