Structured Review

Millipore sp1 antibody
Increased IL-21R expression correlates with increased <t>SP1</t> binding to the IL-21R promoter in RA. (A) Total B cells were isolated from whole blood from RA patients and controls. SP1 binding to the IL21R promoter region and negative control IgG binding were determined using ChIP-qPCR analysis. The results are presented relative to input DNA. Significance was assessed using the student's t -test; n = 4 [Control (black circle), RA High (gray triangle)] n = 3 [RA Low (white square)]. ** p
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1) Product Images from "Increased Binding of Specificity Protein 1 to the IL21R Promoter in B Cells Results in Enhanced B Cell Responses in Rheumatoid Arthritis"

Article Title: Increased Binding of Specificity Protein 1 to the IL21R Promoter in B Cells Results in Enhanced B Cell Responses in Rheumatoid Arthritis

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01978

Increased IL-21R expression correlates with increased SP1 binding to the IL-21R promoter in RA. (A) Total B cells were isolated from whole blood from RA patients and controls. SP1 binding to the IL21R promoter region and negative control IgG binding were determined using ChIP-qPCR analysis. The results are presented relative to input DNA. Significance was assessed using the student's t -test; n = 4 [Control (black circle), RA High (gray triangle)] n = 3 [RA Low (white square)]. ** p
Figure Legend Snippet: Increased IL-21R expression correlates with increased SP1 binding to the IL-21R promoter in RA. (A) Total B cells were isolated from whole blood from RA patients and controls. SP1 binding to the IL21R promoter region and negative control IgG binding were determined using ChIP-qPCR analysis. The results are presented relative to input DNA. Significance was assessed using the student's t -test; n = 4 [Control (black circle), RA High (gray triangle)] n = 3 [RA Low (white square)]. ** p

Techniques Used: Expressing, Binding Assay, Isolation, Negative Control, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Increased SP1 expression correlates with increased IL-21R expression in memory B cells in RA subjects (A) SP1 protein levels were assessed by flow cytometry in control ( n = 6, black circles), RA low IL-21R expressers ( n = 6; open squares) and RA high IL-21R expressers ( n = 5; gray triangles) in CD20 + and memory B cells (CD20 + CD38 − CD24 + ) (left). SP1 protein levels from left were correlated with IL-21R protein expression in total B cells in RA and control subjects ( n = 17) (right). (B) Representative histogram of mRNA levels from a no probe negative control, SP1 and positive control probe, RPL13A . (C) (left) SP1 mRNA levels were determined in total memory B cells in controls ( n = 7, black circles), RA low IL-21R expressers ( n = 7; open squares) and RA high IL-21R expressers ( n = 6; gray triangles). (right) SP1 mRNA levels from left were correlated with IL-21R protein expression in memory B cells in RA and control subjects combined ( n = 20). Significance was determined using Mann Whitney U tests (to compare RA-IL-21 high to controls and RA-IL-21 low ) and correlations were assessed with Pearson correlations.
Figure Legend Snippet: Increased SP1 expression correlates with increased IL-21R expression in memory B cells in RA subjects (A) SP1 protein levels were assessed by flow cytometry in control ( n = 6, black circles), RA low IL-21R expressers ( n = 6; open squares) and RA high IL-21R expressers ( n = 5; gray triangles) in CD20 + and memory B cells (CD20 + CD38 − CD24 + ) (left). SP1 protein levels from left were correlated with IL-21R protein expression in total B cells in RA and control subjects ( n = 17) (right). (B) Representative histogram of mRNA levels from a no probe negative control, SP1 and positive control probe, RPL13A . (C) (left) SP1 mRNA levels were determined in total memory B cells in controls ( n = 7, black circles), RA low IL-21R expressers ( n = 7; open squares) and RA high IL-21R expressers ( n = 6; gray triangles). (right) SP1 mRNA levels from left were correlated with IL-21R protein expression in memory B cells in RA and control subjects combined ( n = 20). Significance was determined using Mann Whitney U tests (to compare RA-IL-21 high to controls and RA-IL-21 low ) and correlations were assessed with Pearson correlations.

Techniques Used: Expressing, Flow Cytometry, Cytometry, Negative Control, Positive Control, MANN-WHITNEY

2) Product Images from "Inhibition of Notch1 signaling overcomes resistance to the death ligand Trail by specificity protein 1-dependent upregulation of death receptor 5"

Article Title: Inhibition of Notch1 signaling overcomes resistance to the death ligand Trail by specificity protein 1-dependent upregulation of death receptor 5

Journal: Cell Death & Disease

doi: 10.1038/cddis.2015.261

Notch1 regulates DR5 via the transcription factor Sp1. ( a and b ) U251MG cells transduced with control-AdV or Notch1sh-AdV were transfected with Sp1 siRNA oligonucleotides 24 h post transduction. ( a ) Immunoblot analysis for Notch1, Sp1, and DR5 72 h post transduction. ( b ) Quantitative real-time PCR analysis of Notch1 and DR5 mRNA expression 72 h post transduction. Expression data were normalized to internal 18 S rRNA expression ( n =3, mean±S.D.). P- values were determined by t -test. ( c ) U251MG cells transduced with control-AdV or Notch1sh-AdV were transfected with Sp1 siRNA oligonucleotides 24 h post transduction. Amount of DR5 located at the cell membrane was determined by flow cytometry 48 h post transfection using a FITC-labeled anti-DR5 antibody (left panel) and a FITC-labeled IgG as a control (right panel). ( d ) Cytoplasmic and nuclear levels of Sp1 remain stable following Notch1 knockdown. Immunoblot analysis for Notch1 and Sp1 in cytoplasmic and nuclear lysate fractions from wild type, control-AdV and Notch1sh-AdV-transduced U251MG cells 72 h post transduction. ( e ) Binding of Sp1 to the DR5 promoter is strongly enhanced following Notch1 downregulation. ChIP analysis of Sp1-binding to the DR5 promoter in U251MG cells transduced with control-AdV or Notch1sh-AdV (72 h) ( n =2; mean±S.D.). Rabbit IgG was used as a control for unspecific binding. ( f ) Relative DR5 promoter activity measured in U251MG cells transduced with control-AdV or Notch1sh-AdV using luciferase reporter constructs containing either the full-length DR5 promoter (−198 bp) or the DR5 promoter with mutated Sp1-binding site 1 (Sp1 BS1mut) and mutated Sp1-binding site 2 (Sp1 BS2mut), respectively ( n =3; mean±S.D.). P -values were determined by t -test. ND, not detected
Figure Legend Snippet: Notch1 regulates DR5 via the transcription factor Sp1. ( a and b ) U251MG cells transduced with control-AdV or Notch1sh-AdV were transfected with Sp1 siRNA oligonucleotides 24 h post transduction. ( a ) Immunoblot analysis for Notch1, Sp1, and DR5 72 h post transduction. ( b ) Quantitative real-time PCR analysis of Notch1 and DR5 mRNA expression 72 h post transduction. Expression data were normalized to internal 18 S rRNA expression ( n =3, mean±S.D.). P- values were determined by t -test. ( c ) U251MG cells transduced with control-AdV or Notch1sh-AdV were transfected with Sp1 siRNA oligonucleotides 24 h post transduction. Amount of DR5 located at the cell membrane was determined by flow cytometry 48 h post transfection using a FITC-labeled anti-DR5 antibody (left panel) and a FITC-labeled IgG as a control (right panel). ( d ) Cytoplasmic and nuclear levels of Sp1 remain stable following Notch1 knockdown. Immunoblot analysis for Notch1 and Sp1 in cytoplasmic and nuclear lysate fractions from wild type, control-AdV and Notch1sh-AdV-transduced U251MG cells 72 h post transduction. ( e ) Binding of Sp1 to the DR5 promoter is strongly enhanced following Notch1 downregulation. ChIP analysis of Sp1-binding to the DR5 promoter in U251MG cells transduced with control-AdV or Notch1sh-AdV (72 h) ( n =2; mean±S.D.). Rabbit IgG was used as a control for unspecific binding. ( f ) Relative DR5 promoter activity measured in U251MG cells transduced with control-AdV or Notch1sh-AdV using luciferase reporter constructs containing either the full-length DR5 promoter (−198 bp) or the DR5 promoter with mutated Sp1-binding site 1 (Sp1 BS1mut) and mutated Sp1-binding site 2 (Sp1 BS2mut), respectively ( n =3; mean±S.D.). P -values were determined by t -test. ND, not detected

Techniques Used: Transduction, Transfection, Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry, Cytometry, Labeling, Binding Assay, Chromatin Immunoprecipitation, Activity Assay, Luciferase, Construct

3) Product Images from "EHMT2 epigenetically suppresses Wnt signaling and is a potential target in embryonal rhabdomyosarcoma"

Article Title: EHMT2 epigenetically suppresses Wnt signaling and is a potential target in embryonal rhabdomyosarcoma

Journal: eLife

doi: 10.7554/eLife.57683

EHMT2 regulates Sp1 and p300 occupancy at the DKK1 promoter. ( A ) Top DNA motifs at EHMT2-predicted binding sites by HOMER analysis. ( B and C ) ChIP-PCR analysis showed decrease in p300 occupancy and H3K9ac enrichment at the DKK1 promoter in shEHMT2 RD cells as compared to control (n = 2). Error bars indicate the mean ± SD. ( D ) Proximity ligation assay (PLA) was done to examine Sp1 and p300 interaction in control and 2.5 μM of UNC0642-treated RD cells. Sp1–p300 interaction showed a significant decrease upon UNC0642 treatment. Images were captured using confocal microscopy. Data shown is representative of two independent experiments. Error bars indicate the mean ± SD. ( E ) PLA was done to examine Sp1 and p300 interaction in control and 2.5 μM of UNC0642-treated human skeletal muscle myoblast cells. Sp1-p300 interaction showed a significant decrease upon UNC0642 treatment. Images were captured using confocal microscopy. Data shown is representative of two independent experiments. Error bars indicate the mean ± SD. ( F ) PLA was used to determine EHMT2-Sp1 interaction which did not change upon UNC0642 treatment. Data shown is representative of two independent experiments. Error bars indicate the mean ± SD. ( G ) EHMT2-p300 interaction was not significantly altered by UNC0642 treatment. Data shown is representative of two independent experiments. Error bars indicate the mean ± SD. ( H ) Single antibody controls for p300, EHMT2, and Sp1 were done for PLA in RD18 cells. In ( B–G ) the bar graphs are plotted for one biological representative each with three technical replicates of two biological experiments. Statistical significance in ( B–G ) was calculated as unpaired two-tailed t -test. **p≤0.001, ns = not significant.
Figure Legend Snippet: EHMT2 regulates Sp1 and p300 occupancy at the DKK1 promoter. ( A ) Top DNA motifs at EHMT2-predicted binding sites by HOMER analysis. ( B and C ) ChIP-PCR analysis showed decrease in p300 occupancy and H3K9ac enrichment at the DKK1 promoter in shEHMT2 RD cells as compared to control (n = 2). Error bars indicate the mean ± SD. ( D ) Proximity ligation assay (PLA) was done to examine Sp1 and p300 interaction in control and 2.5 μM of UNC0642-treated RD cells. Sp1–p300 interaction showed a significant decrease upon UNC0642 treatment. Images were captured using confocal microscopy. Data shown is representative of two independent experiments. Error bars indicate the mean ± SD. ( E ) PLA was done to examine Sp1 and p300 interaction in control and 2.5 μM of UNC0642-treated human skeletal muscle myoblast cells. Sp1-p300 interaction showed a significant decrease upon UNC0642 treatment. Images were captured using confocal microscopy. Data shown is representative of two independent experiments. Error bars indicate the mean ± SD. ( F ) PLA was used to determine EHMT2-Sp1 interaction which did not change upon UNC0642 treatment. Data shown is representative of two independent experiments. Error bars indicate the mean ± SD. ( G ) EHMT2-p300 interaction was not significantly altered by UNC0642 treatment. Data shown is representative of two independent experiments. Error bars indicate the mean ± SD. ( H ) Single antibody controls for p300, EHMT2, and Sp1 were done for PLA in RD18 cells. In ( B–G ) the bar graphs are plotted for one biological representative each with three technical replicates of two biological experiments. Statistical significance in ( B–G ) was calculated as unpaired two-tailed t -test. **p≤0.001, ns = not significant.

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Proximity Ligation Assay, Confocal Microscopy, Two Tailed Test

4) Product Images from "Specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 are non-oncogene addiction genes in cancer cells"

Article Title: Specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 are non-oncogene addiction genes in cancer cells

Journal: Oncotarget

doi: 10.18632/oncotarget.7925

Changes in expression of specific genes after Sp knockdown in Panc1 cells Panc1 cells were transfected with siSp1, siSp3 or siSp4, and real time PCR analysis was used to determine changes in expression of A. RRM2 and AURKA, B. TXNIP and CBX7, C. CASP3 and SPRY2, D. HMOX1 and ISG15, and E. Sp1, Sp3 and Sp4. Results are expressed as means ± SE for at least 3 replicates for each treatment group, and significantly (p
Figure Legend Snippet: Changes in expression of specific genes after Sp knockdown in Panc1 cells Panc1 cells were transfected with siSp1, siSp3 or siSp4, and real time PCR analysis was used to determine changes in expression of A. RRM2 and AURKA, B. TXNIP and CBX7, C. CASP3 and SPRY2, D. HMOX1 and ISG15, and E. Sp1, Sp3 and Sp4. Results are expressed as means ± SE for at least 3 replicates for each treatment group, and significantly (p

Techniques Used: Expressing, Transfection, Real-time Polymerase Chain Reaction

Functional effects of Sp1, Sp3 and Sp4 in A549, MiaPaCa2, SW480, 786-O, SKBR3, MDA-MB-231, Panc1 and L3.6pL cancer cell lines Cells were transfected with siSp1, siSp3 and siSp4 and effects on cell proliferation A. Annexin V staining B. and invasion in a Boyden chamber assay C. were determined as described in the Materials and Methods. Results are expressed as means ± SE for at least 3 biological replicates for each determination, and significant (p
Figure Legend Snippet: Functional effects of Sp1, Sp3 and Sp4 in A549, MiaPaCa2, SW480, 786-O, SKBR3, MDA-MB-231, Panc1 and L3.6pL cancer cell lines Cells were transfected with siSp1, siSp3 and siSp4 and effects on cell proliferation A. Annexin V staining B. and invasion in a Boyden chamber assay C. were determined as described in the Materials and Methods. Results are expressed as means ± SE for at least 3 biological replicates for each determination, and significant (p

Techniques Used: Functional Assay, Multiple Displacement Amplification, Transfection, Staining, Boyden Chamber Assay

Analysis of changes in gene expression after knockdown of Sp1, Sp3 and Sp4 in Panc1 cells A. Panc1 cells were transfected with siSp1, siSp3 or siSp4, and changes in gene expression were determined using Human HT-12 V4 expression bead chip arrays. The overlap of total genes B. and growth inhibition C. cell death D. and inhibition of migration/invasion E. genes coregulated by Sp1/Sp3, Sp1, Sp4 and Sp3/Sp4 in Panc1 cells after RNAi was determined by IPA.
Figure Legend Snippet: Analysis of changes in gene expression after knockdown of Sp1, Sp3 and Sp4 in Panc1 cells A. Panc1 cells were transfected with siSp1, siSp3 or siSp4, and changes in gene expression were determined using Human HT-12 V4 expression bead chip arrays. The overlap of total genes B. and growth inhibition C. cell death D. and inhibition of migration/invasion E. genes coregulated by Sp1/Sp3, Sp1, Sp4 and Sp3/Sp4 in Panc1 cells after RNAi was determined by IPA.

Techniques Used: Expressing, Transfection, Chromatin Immunoprecipitation, Inhibition, Migration, Indirect Immunoperoxidase Assay

5) Product Images from "Mitochondria-Localized Glutamic Acid-Rich Protein (MGARP) Gene Transcription Is Regulated by Sp1"

Article Title: Mitochondria-Localized Glutamic Acid-Rich Protein (MGARP) Gene Transcription Is Regulated by Sp1

Journal: PLoS ONE

doi: 10.1371/journal.pone.0050053

EMSA test indicates that Sp1 directly binds to the GC-boxes of the MGARP promoter. For the EMSA analysis, nuclear extracts (Nu) from HEK-293T or Y1 cells were incubated with Biotin-labeled oligonucleotides (Biotin-probe) spanning the GC-rich region (BOX1) of the MGARP promoter (−3 kb). Competition reactions were performed with 200X of unlabeled cold competitor (cold), 200X of mutated-labeled competitors (mu) or Sp1 antibody (2 µg). The following cell lines were used: non-transfected or Sp1-overexpressed HEK-293T cells (A), non-transfected, Sp1-overexpressed, or 630-RNAi transfected HEK-293T cells (B), and Y1 cells (C).
Figure Legend Snippet: EMSA test indicates that Sp1 directly binds to the GC-boxes of the MGARP promoter. For the EMSA analysis, nuclear extracts (Nu) from HEK-293T or Y1 cells were incubated with Biotin-labeled oligonucleotides (Biotin-probe) spanning the GC-rich region (BOX1) of the MGARP promoter (−3 kb). Competition reactions were performed with 200X of unlabeled cold competitor (cold), 200X of mutated-labeled competitors (mu) or Sp1 antibody (2 µg). The following cell lines were used: non-transfected or Sp1-overexpressed HEK-293T cells (A), non-transfected, Sp1-overexpressed, or 630-RNAi transfected HEK-293T cells (B), and Y1 cells (C).

Techniques Used: Incubation, Labeling, Transfection

ChIP analysis indicates that Sp1 binds to MGARP promoter in vivo . ChIP was performed as described in the Materials and Methods . HEK-293T cells and antibodies for RNA polymerase II (Pol II) and Sp1 were used, with IgG as control. The immunoprecipitated chromatin was amplified by PCR with primers specific for the GC-rich region (BOX1 2) of the MGARP promoter (−3 kb), with GAPDH locus as control. M: DNA Marker.
Figure Legend Snippet: ChIP analysis indicates that Sp1 binds to MGARP promoter in vivo . ChIP was performed as described in the Materials and Methods . HEK-293T cells and antibodies for RNA polymerase II (Pol II) and Sp1 were used, with IgG as control. The immunoprecipitated chromatin was amplified by PCR with primers specific for the GC-rich region (BOX1 2) of the MGARP promoter (−3 kb), with GAPDH locus as control. M: DNA Marker.

Techniques Used: Chromatin Immunoprecipitation, In Vivo, Immunoprecipitation, Amplification, Polymerase Chain Reaction, Marker

ERα up-regulates the transcription of the MGARP promoter and acts in synergy with Sp1 to activate MGARP transcriptional activity. A. pGL3-(−3 kb) reporter and different doses of ERα expression plasmid were co-transfected into HEK-293T cells to determine the dose-dependent manner of ERα in regulating the MGARP promoter by luciferase assay. B. The functional synergy between Sp1 and ERα was determined by cotransfection of the full-length MGARP promoter (−3 kb) or various promoter truncates with or without Sp1 plasmids for Luc assay as indicated. C. The synergystic transactivation activity of ERα and Sp1 under the stimulation of estrogens. The HEK-293T cells were treated with or without 10 nM of estradiol (E2) for 24 hours post transfection of pGL3-(−3 kb) and ERα. Subsequently, the Luc assay was performed at 72 hours post transfection. D. Knockdown of Sp1 diminishes the activation function of ERα on MGARP promoter. HEK-293T cells were co-transfected with pGL3-(−3 kb) reporter and ERα, together with Sp1-specific RNAi (630-RNAi or 1722-RNAi) or control RNAi, in the absence or presence of exogenous Sp1. *** represents p
Figure Legend Snippet: ERα up-regulates the transcription of the MGARP promoter and acts in synergy with Sp1 to activate MGARP transcriptional activity. A. pGL3-(−3 kb) reporter and different doses of ERα expression plasmid were co-transfected into HEK-293T cells to determine the dose-dependent manner of ERα in regulating the MGARP promoter by luciferase assay. B. The functional synergy between Sp1 and ERα was determined by cotransfection of the full-length MGARP promoter (−3 kb) or various promoter truncates with or without Sp1 plasmids for Luc assay as indicated. C. The synergystic transactivation activity of ERα and Sp1 under the stimulation of estrogens. The HEK-293T cells were treated with or without 10 nM of estradiol (E2) for 24 hours post transfection of pGL3-(−3 kb) and ERα. Subsequently, the Luc assay was performed at 72 hours post transfection. D. Knockdown of Sp1 diminishes the activation function of ERα on MGARP promoter. HEK-293T cells were co-transfected with pGL3-(−3 kb) reporter and ERα, together with Sp1-specific RNAi (630-RNAi or 1722-RNAi) or control RNAi, in the absence or presence of exogenous Sp1. *** represents p

Techniques Used: Activity Assay, Expressing, Plasmid Preparation, Transfection, Luciferase, Functional Assay, Cotransfection, Activation Assay

GC-box1 plays a major role in MGARP promoter activation and both GC-boxes are required for full transactivation. The Luc reporters driven by the full-length MGARP promoter (−3 kb) were transfected into HEK-293T cells, as compared to various promoter truncates either missing the GC-Boxes or expressing the GC-Boxes alone, without or with co-transfection of Sp1 plasmids ( 10 ng ) as indicated. Luc activity was examined at 72 hours post transfection. *** represents p
Figure Legend Snippet: GC-box1 plays a major role in MGARP promoter activation and both GC-boxes are required for full transactivation. The Luc reporters driven by the full-length MGARP promoter (−3 kb) were transfected into HEK-293T cells, as compared to various promoter truncates either missing the GC-Boxes or expressing the GC-Boxes alone, without or with co-transfection of Sp1 plasmids ( 10 ng ) as indicated. Luc activity was examined at 72 hours post transfection. *** represents p

Techniques Used: Activation Assay, Transfection, Expressing, Cotransfection, Activity Assay

MGARP promoter activation is mediated by Sp1. A. Luc assay shows that Sp1 mediates the MGARP promoter activity in a dose-dependent manner. HEK-293T cells were co-transfected with pGL3-(−3 kb) and the increasing doses of Sp1 plasmids for Luc assay. B. Similarly, the pDsRed-MGARP(−3 kb) reporter and several doses of Sp1 plasmids were co-transfected into HEK-293T cells to examine the expression of red fluorescent protein at 72 hours post transfection. C. Western blotting shows that knockdown of Sp1 with Sp1-specific RNAi (630-RNAi and 1722-RNAi) reduces the expression of both endogenous and exogenous Sp1. The scramble-RNAi and RNAi targeting GFP were used as control. D. Luc assays to determine the effect of Sp1 knockdown on MGARP promoter activity. *** represents p
Figure Legend Snippet: MGARP promoter activation is mediated by Sp1. A. Luc assay shows that Sp1 mediates the MGARP promoter activity in a dose-dependent manner. HEK-293T cells were co-transfected with pGL3-(−3 kb) and the increasing doses of Sp1 plasmids for Luc assay. B. Similarly, the pDsRed-MGARP(−3 kb) reporter and several doses of Sp1 plasmids were co-transfected into HEK-293T cells to examine the expression of red fluorescent protein at 72 hours post transfection. C. Western blotting shows that knockdown of Sp1 with Sp1-specific RNAi (630-RNAi and 1722-RNAi) reduces the expression of both endogenous and exogenous Sp1. The scramble-RNAi and RNAi targeting GFP were used as control. D. Luc assays to determine the effect of Sp1 knockdown on MGARP promoter activity. *** represents p

Techniques Used: Activation Assay, Activity Assay, Transfection, Expressing, Western Blot

6) Product Images from "Suppression of MicroRNA 200 Family Expression by Oncogenic KRAS Activation Promotes Cell Survival and Epithelial-Mesenchymal Transition in KRAS-Driven Cancer"

Article Title: Suppression of MicroRNA 200 Family Expression by Oncogenic KRAS Activation Promotes Cell Survival and Epithelial-Mesenchymal Transition in KRAS-Driven Cancer

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00079-16

KRAS suppresses mir-200 family expression through its downstream effectors JUN and SP1. (A) Downstream signaling pathways and effectors activated by KRAS. (B) Screening for effectors responsible for the suppressive function on mir-200 expression with
Figure Legend Snippet: KRAS suppresses mir-200 family expression through its downstream effectors JUN and SP1. (A) Downstream signaling pathways and effectors activated by KRAS. (B) Screening for effectors responsible for the suppressive function on mir-200 expression with

Techniques Used: Expressing

The signaling network connecting RAS and mir-200 regulates apoptosis and EMT in cancer cells. Through the signaling pathway RAF/MEK/ERK and transcription factors JUN and SP1, RAS suppresses mir-200 and hence stabilizes the expression level of BCL2 and
Figure Legend Snippet: The signaling network connecting RAS and mir-200 regulates apoptosis and EMT in cancer cells. Through the signaling pathway RAF/MEK/ERK and transcription factors JUN and SP1, RAS suppresses mir-200 and hence stabilizes the expression level of BCL2 and

Techniques Used: Expressing

7) Product Images from ""

Article Title:

Journal: Molecular Pharmacology

doi: 10.1124/mol.110.064451

CDDO-Me down-regulates Sp proteins in a proteosome-independent manner. CDDO-Me decreases Sp protein expression in Panc1 (A), Panc28 (B), and L3.6pL (C). Cells were with DMSO, CDDO (1.0, 2.5, or 5.0 μM), or CDDO-Me (0.5, 1.0, or 1.25 μM) for 24 h, and whole-cell lysates were analyzed for Sp1, Sp3, and Sp4 by Western blot analysis as described under Materials and Methods . D, proteosome-independent down-regulation of Sp proteins by CDDO-Me. Cells were treated with DMSO and CDDO-Me (1.0 μM) in the presence or absence of proteasome inhibitor MG132 (10 μM), and the effects on Sp protein degradation were determined after treatment for 24 h by Western blot as described under Materials and Methods . β-Actin served as a loading control.
Figure Legend Snippet: CDDO-Me down-regulates Sp proteins in a proteosome-independent manner. CDDO-Me decreases Sp protein expression in Panc1 (A), Panc28 (B), and L3.6pL (C). Cells were with DMSO, CDDO (1.0, 2.5, or 5.0 μM), or CDDO-Me (0.5, 1.0, or 1.25 μM) for 24 h, and whole-cell lysates were analyzed for Sp1, Sp3, and Sp4 by Western blot analysis as described under Materials and Methods . D, proteosome-independent down-regulation of Sp proteins by CDDO-Me. Cells were treated with DMSO and CDDO-Me (1.0 μM) in the presence or absence of proteasome inhibitor MG132 (10 μM), and the effects on Sp protein degradation were determined after treatment for 24 h by Western blot as described under Materials and Methods . β-Actin served as a loading control.

Techniques Used: Expressing, Western Blot

8) Product Images from "Inhibition of NF?B and Pancreatic Cancer Cell and Tumor Growth by Curcumin Is Dependent on Specificity Protein Down-regulation *"

Article Title: Inhibition of NF?B and Pancreatic Cancer Cell and Tumor Growth by Curcumin Is Dependent on Specificity Protein Down-regulation *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.095240

Time course effects of curcumin on Sp1, Sp3, Sp4, p65 and p50, and ROS. Panc28 ( A ) and L3.6pL ( B ) cells were treated with DMSO (0 time) or 50 μ m curcumin for different times over a 24-h period and whole cell lysates were analyzed by Western blots
Figure Legend Snippet: Time course effects of curcumin on Sp1, Sp3, Sp4, p65 and p50, and ROS. Panc28 ( A ) and L3.6pL ( B ) cells were treated with DMSO (0 time) or 50 μ m curcumin for different times over a 24-h period and whole cell lysates were analyzed by Western blots

Techniques Used: Western Blot

9) Product Images from "Quercetin enhances ABCA1 expression and cholesterol efflux through a p38-dependent pathway in macrophages"

Article Title: Quercetin enhances ABCA1 expression and cholesterol efflux through a p38-dependent pathway in macrophages

Journal: Journal of Lipid Research

doi: 10.1194/jlr.M024471

A model describing the mechanisms of quercetin-induced cholesterol efflux in macrophages. Quercetin elicits the TAK1-MKK3/6 signaling cascade to activate p38. Activated p38 subsequently increases the binding of Sp1 and LXRα to the ABCA1 promoter, which in turn enhances the expression of ABCA1 as well as cholesterol efflux from macrophages.
Figure Legend Snippet: A model describing the mechanisms of quercetin-induced cholesterol efflux in macrophages. Quercetin elicits the TAK1-MKK3/6 signaling cascade to activate p38. Activated p38 subsequently increases the binding of Sp1 and LXRα to the ABCA1 promoter, which in turn enhances the expression of ABCA1 as well as cholesterol efflux from macrophages.

Techniques Used: Binding Assay, Expressing

Sp1 and LXR binding sites are quercetin-responsive elements within the ABCA1 promoter. A: Six mutation constructs of ABCA1 promoter in the potential Sp1a, E-box, AP1, Sp1b, and LXR recognition sites were generated using site-directed mutatgenesis. RAW264.7 macrophages were transiently cotransfected with 1 μg of indicated mutant constructs and 0.5 μg of pCMV β-galactosidase expression plasmid, and then treated with 100 μM quercetin (Q100) for 24 h. Luciferase activity was normalized with β-galactosidase activity and expressed as fold changes in luciferase activity compared with those of the control. The level of luciferase activity without quercetin treatment was given the value of 1. WT-250, −250/−1 region of ABCA1 promoter. mSp1b/mLXR, double mutations at Sp1b and LXR sites. B: Cells were treated with quercetin for 3 h, and nuclear extracts were harvested. The nuclear expression levels of Sp1 and LXR were analyzed by Western blot analysis. The arrow indicates Sp1 band. B23 was used as nuclear loading control, and α-tubulin was used to exclude cytosolic contamination. The level of Sp1 or LXR without quercetin treatment was given the value of 1. C: ChIP assays were performed to observe the binding of Sp1 and LXRα to the ABCA1 promoter in quercetin-treated cells as described in Materials and Methods. Nonimmune IgG was used as the negative control. The results of the ChIP assays were evaluated by PCR and gel electrophoresis. Values are quantified by densitometer and expressed as ratio relative to inputs. Results are mean ± SEM (n = 3–4). ** P
Figure Legend Snippet: Sp1 and LXR binding sites are quercetin-responsive elements within the ABCA1 promoter. A: Six mutation constructs of ABCA1 promoter in the potential Sp1a, E-box, AP1, Sp1b, and LXR recognition sites were generated using site-directed mutatgenesis. RAW264.7 macrophages were transiently cotransfected with 1 μg of indicated mutant constructs and 0.5 μg of pCMV β-galactosidase expression plasmid, and then treated with 100 μM quercetin (Q100) for 24 h. Luciferase activity was normalized with β-galactosidase activity and expressed as fold changes in luciferase activity compared with those of the control. The level of luciferase activity without quercetin treatment was given the value of 1. WT-250, −250/−1 region of ABCA1 promoter. mSp1b/mLXR, double mutations at Sp1b and LXR sites. B: Cells were treated with quercetin for 3 h, and nuclear extracts were harvested. The nuclear expression levels of Sp1 and LXR were analyzed by Western blot analysis. The arrow indicates Sp1 band. B23 was used as nuclear loading control, and α-tubulin was used to exclude cytosolic contamination. The level of Sp1 or LXR without quercetin treatment was given the value of 1. C: ChIP assays were performed to observe the binding of Sp1 and LXRα to the ABCA1 promoter in quercetin-treated cells as described in Materials and Methods. Nonimmune IgG was used as the negative control. The results of the ChIP assays were evaluated by PCR and gel electrophoresis. Values are quantified by densitometer and expressed as ratio relative to inputs. Results are mean ± SEM (n = 3–4). ** P

Techniques Used: Binding Assay, Mutagenesis, Construct, Generated, Expressing, Plasmid Preparation, Luciferase, Activity Assay, Western Blot, Chromatin Immunoprecipitation, Negative Control, Polymerase Chain Reaction, Nucleic Acid Electrophoresis

Effect of p38 knockdown by shRNA on the attenuation of Sp1 and LXRα binding to the ABCA1 promoter. A: Parental cells or p38 knockdown cells were transiently cotransfected with 0.5 μg of WT-250 construct, 0.5 μg of pRcCMV vector, and 0.5 μg of pCMV β-galactosidase plasmid. For p38 overexpression, p38 knockdown cells were transiently cotransfected with 0.5 μg of WT-250 construct, 0.5 μg of pRcCMV-p38 plasmid, and 0.5 μg of pCMV β-galactosidase plasmid. After transfection, cells were treated with 100 μM quercetin (Q100) for 24 h. Luciferase activity was normalized with β-galactosidase activity and expressed as fold changes in luciferase activity compared with respective control groups. The level of luciferase activity without quercetin treatment was given the value of 1. WT-250, −250/−1 region of ABCA1 promoter. B: ChIP assays were performed to observe the binding of Sp1 and LXRα to the ABCA1 promoter in p38 knockdown cells as described in Materials and Methods. Nonimmune IgG was used as the negative control. The results of ChIP assays were evaluated by PCR and gel electrophoresis. Values are quantified by densitometer and expressed as ratio relative to inputs. Bars are mean ± SEM (n = 3). ** P
Figure Legend Snippet: Effect of p38 knockdown by shRNA on the attenuation of Sp1 and LXRα binding to the ABCA1 promoter. A: Parental cells or p38 knockdown cells were transiently cotransfected with 0.5 μg of WT-250 construct, 0.5 μg of pRcCMV vector, and 0.5 μg of pCMV β-galactosidase plasmid. For p38 overexpression, p38 knockdown cells were transiently cotransfected with 0.5 μg of WT-250 construct, 0.5 μg of pRcCMV-p38 plasmid, and 0.5 μg of pCMV β-galactosidase plasmid. After transfection, cells were treated with 100 μM quercetin (Q100) for 24 h. Luciferase activity was normalized with β-galactosidase activity and expressed as fold changes in luciferase activity compared with respective control groups. The level of luciferase activity without quercetin treatment was given the value of 1. WT-250, −250/−1 region of ABCA1 promoter. B: ChIP assays were performed to observe the binding of Sp1 and LXRα to the ABCA1 promoter in p38 knockdown cells as described in Materials and Methods. Nonimmune IgG was used as the negative control. The results of ChIP assays were evaluated by PCR and gel electrophoresis. Values are quantified by densitometer and expressed as ratio relative to inputs. Bars are mean ± SEM (n = 3). ** P

Techniques Used: shRNA, Binding Assay, Construct, Plasmid Preparation, Over Expression, Transfection, Luciferase, Activity Assay, Chromatin Immunoprecipitation, Negative Control, Polymerase Chain Reaction, Nucleic Acid Electrophoresis

10) Product Images from "Increased Binding of Specificity Protein 1 to the IL21R Promoter in B Cells Results in Enhanced B Cell Responses in Rheumatoid Arthritis"

Article Title: Increased Binding of Specificity Protein 1 to the IL21R Promoter in B Cells Results in Enhanced B Cell Responses in Rheumatoid Arthritis

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01978

Increased IL-21R expression correlates with increased SP1 binding to the IL-21R promoter in RA. (A) Total B cells were isolated from whole blood from RA patients and controls. SP1 binding to the IL21R promoter region and negative control IgG binding were determined using ChIP-qPCR analysis. The results are presented relative to input DNA. Significance was assessed using the student's t -test; n = 4 [Control (black circle), RA High (gray triangle)] n = 3 [RA Low (white square)]. ** p
Figure Legend Snippet: Increased IL-21R expression correlates with increased SP1 binding to the IL-21R promoter in RA. (A) Total B cells were isolated from whole blood from RA patients and controls. SP1 binding to the IL21R promoter region and negative control IgG binding were determined using ChIP-qPCR analysis. The results are presented relative to input DNA. Significance was assessed using the student's t -test; n = 4 [Control (black circle), RA High (gray triangle)] n = 3 [RA Low (white square)]. ** p

Techniques Used: Expressing, Binding Assay, Isolation, Negative Control, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Increased SP1 expression correlates with increased IL-21R expression in memory B cells in RA subjects (A) SP1 protein levels were assessed by flow cytometry in control ( n = 6, black circles), RA low IL-21R expressers ( n = 6; open squares) and RA high IL-21R expressers ( n = 5; gray triangles) in CD20 + and memory B cells (CD20 + CD38 − CD24 + ) (left). SP1 protein levels from left were correlated with IL-21R protein expression in total B cells in RA and control subjects ( n = 17) (right). (B) Representative histogram of mRNA levels from a no probe negative control, SP1 and positive control probe, RPL13A . (C) (left) SP1 mRNA levels were determined in total memory B cells in controls ( n = 7, black circles), RA low IL-21R expressers ( n = 7; open squares) and RA high IL-21R expressers ( n = 6; gray triangles). (right) SP1 mRNA levels from left were correlated with IL-21R protein expression in memory B cells in RA and control subjects combined ( n = 20). Significance was determined using Mann Whitney U tests (to compare RA-IL-21 high to controls and RA-IL-21 low ) and correlations were assessed with Pearson correlations.
Figure Legend Snippet: Increased SP1 expression correlates with increased IL-21R expression in memory B cells in RA subjects (A) SP1 protein levels were assessed by flow cytometry in control ( n = 6, black circles), RA low IL-21R expressers ( n = 6; open squares) and RA high IL-21R expressers ( n = 5; gray triangles) in CD20 + and memory B cells (CD20 + CD38 − CD24 + ) (left). SP1 protein levels from left were correlated with IL-21R protein expression in total B cells in RA and control subjects ( n = 17) (right). (B) Representative histogram of mRNA levels from a no probe negative control, SP1 and positive control probe, RPL13A . (C) (left) SP1 mRNA levels were determined in total memory B cells in controls ( n = 7, black circles), RA low IL-21R expressers ( n = 7; open squares) and RA high IL-21R expressers ( n = 6; gray triangles). (right) SP1 mRNA levels from left were correlated with IL-21R protein expression in memory B cells in RA and control subjects combined ( n = 20). Significance was determined using Mann Whitney U tests (to compare RA-IL-21 high to controls and RA-IL-21 low ) and correlations were assessed with Pearson correlations.

Techniques Used: Expressing, Flow Cytometry, Cytometry, Negative Control, Positive Control, MANN-WHITNEY

11) Product Images from "Tumor Necrosis Factor Alpha Regulates Skeletal Myogenesis by Inhibiting SP1 Interaction with cis-Acting Regulatory Elements within the Fbxl2 Gene Promoter"

Article Title: Tumor Necrosis Factor Alpha Regulates Skeletal Myogenesis by Inhibiting SP1 Interaction with cis-Acting Regulatory Elements within the Fbxl2 Gene Promoter

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00040-20

SP1 binds to the Fbxl2 promoter region to regulate its gene expression during myogenic differentiation. (A) Sp1 mRNA abundance following knockdown of SP1 in C2C12 cells. (B) Fbxl2 mRNA abundance by qPCR after SP1 silencing at 48 h postdifferentiation. (C) Relative luminescence after SP1 depletion in C2C12 cells overexpressing the +240 to −42 Fbxl2 promoter reporter construct. (D and E) SP1 and FBXL2 protein levels following depletion (D) and overexpression (E) of SP1 in C2C12 cells. The data are representative of the results of three independent experiments. (F) C2C12 lysates were immunoprecipitated with SP1 antibody. The precipitated DNA was amplified using specific primers to the proximal Fbxl2 promoter region and measured by qPCR; values are expressed as percentages of DNA in the SP1 fractions compared to input. Shown is a quantification graph for ChIP assays using qPCR data demonstrating significant SP1 binding to a region including the Fbxl2 promoter under differentiation conditions (**, P = 0.002) but not following TNF-α stimulation. The data are representative of the results of two independent experiments. (G) The 371-bp region of the Fbxl2 core promoter was amplified by PCR from SP1 and input lysates and visualized on an agarose gel. (H) Nuclear extracts were isolated from C2C12 myoblasts during proliferation and differentiation at the indicated time points in the presence or absence of TNF-α stimulation and then incubated with a biotin-labeled 25-nucleotide segment that corresponds to the proximal putative SP1 binding element of the human Fbxl2 and mouse Fbxl2 core promoters. An EMSA demonstrated the formation of two DNA-protein complexes during myogenic differentiation, which was decreased in the presence of TNF-α-stimulated cells. The data are representative of the results of two independent experiments. (I) Nuclear SP1 expression in C2C12 cells after differentiation with and without TNF-α. (Bottom) Protein levels and band intensities were quantitated and graphed as shown. (A to C, F, and I) **, P
Figure Legend Snippet: SP1 binds to the Fbxl2 promoter region to regulate its gene expression during myogenic differentiation. (A) Sp1 mRNA abundance following knockdown of SP1 in C2C12 cells. (B) Fbxl2 mRNA abundance by qPCR after SP1 silencing at 48 h postdifferentiation. (C) Relative luminescence after SP1 depletion in C2C12 cells overexpressing the +240 to −42 Fbxl2 promoter reporter construct. (D and E) SP1 and FBXL2 protein levels following depletion (D) and overexpression (E) of SP1 in C2C12 cells. The data are representative of the results of three independent experiments. (F) C2C12 lysates were immunoprecipitated with SP1 antibody. The precipitated DNA was amplified using specific primers to the proximal Fbxl2 promoter region and measured by qPCR; values are expressed as percentages of DNA in the SP1 fractions compared to input. Shown is a quantification graph for ChIP assays using qPCR data demonstrating significant SP1 binding to a region including the Fbxl2 promoter under differentiation conditions (**, P = 0.002) but not following TNF-α stimulation. The data are representative of the results of two independent experiments. (G) The 371-bp region of the Fbxl2 core promoter was amplified by PCR from SP1 and input lysates and visualized on an agarose gel. (H) Nuclear extracts were isolated from C2C12 myoblasts during proliferation and differentiation at the indicated time points in the presence or absence of TNF-α stimulation and then incubated with a biotin-labeled 25-nucleotide segment that corresponds to the proximal putative SP1 binding element of the human Fbxl2 and mouse Fbxl2 core promoters. An EMSA demonstrated the formation of two DNA-protein complexes during myogenic differentiation, which was decreased in the presence of TNF-α-stimulated cells. The data are representative of the results of two independent experiments. (I) Nuclear SP1 expression in C2C12 cells after differentiation with and without TNF-α. (Bottom) Protein levels and band intensities were quantitated and graphed as shown. (A to C, F, and I) **, P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Construct, Over Expression, Immunoprecipitation, Amplification, Chromatin Immunoprecipitation, Binding Assay, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Isolation, Incubation, Labeling

JNK-mediated phosphorylation of SP1 inhibits Fbxl2 transcriptional activity in response to TNF-α. (A) Expression of total and phosphorylated JNK, ERK1/2, p38, and AKT with and without TNF-α treatment in C2C12 cells. (B) Protein densitometry was quantitated and graphed. The P values shown represent significance as indicated by the brackets between bars or that of trend analysis over time as analyzed by ANOVA. (C) Fbxl2 promoter-reporter activity in C2C12 cells treated with chemical inhibitors of JNK, ERK1/2, and p38 (1 to 10 μM) for 24 h. (D) Fbxl2 promoter-reporter activity in C2C12 cells after knockdown of JNK with siRNA for 48 h. (E) Fbxl2 promoter-reporter activity in C2C12 cells pretreated with the JNK inhibitor SP600125 (1 μM) 30 min prior to TNF-α treatment. Cells were harvested at 48 h posttreatment. (F) Fbxl2 promoter-reporter activity in HEK cells transfected with SP1 plasmids encoding phosphorylation-deficient mutants (T278A and T739A) or phosphorylation mimics (T278D and T739D) for 48 h. (G) C2C12 cells were transfected with plasmids expressing V5-tagged SP1 . The cells were fixed and sonicated, and DNA was immunoprecipitated with isotype control (IgG), V5, or STAT-1 antibodies. (Left) Relative enrichment of the Fbxl2 promoter was quantified by qPCR. (Right) Agarose gel of a 193-bp region of the Fbxl2 core promoter amplified by PCR. (H) C2C12 cells were transfected with plasmids expressing V5-tagged SP1 wild type (Wt), SP1-T739A, and SP1-T739D for 48 h. The cells were fixed and sonicated, and DNA was immunoprecipitated (IP) with V5 antibody. (Left) Relative enrichment of the Fbxl2 promoter was quantified by qPCR. (Right) Agarose gel of the 193-bp region of the Fbxl2 core promoter amplified by PCR. ns, not significant ( P > 0.05); *, P
Figure Legend Snippet: JNK-mediated phosphorylation of SP1 inhibits Fbxl2 transcriptional activity in response to TNF-α. (A) Expression of total and phosphorylated JNK, ERK1/2, p38, and AKT with and without TNF-α treatment in C2C12 cells. (B) Protein densitometry was quantitated and graphed. The P values shown represent significance as indicated by the brackets between bars or that of trend analysis over time as analyzed by ANOVA. (C) Fbxl2 promoter-reporter activity in C2C12 cells treated with chemical inhibitors of JNK, ERK1/2, and p38 (1 to 10 μM) for 24 h. (D) Fbxl2 promoter-reporter activity in C2C12 cells after knockdown of JNK with siRNA for 48 h. (E) Fbxl2 promoter-reporter activity in C2C12 cells pretreated with the JNK inhibitor SP600125 (1 μM) 30 min prior to TNF-α treatment. Cells were harvested at 48 h posttreatment. (F) Fbxl2 promoter-reporter activity in HEK cells transfected with SP1 plasmids encoding phosphorylation-deficient mutants (T278A and T739A) or phosphorylation mimics (T278D and T739D) for 48 h. (G) C2C12 cells were transfected with plasmids expressing V5-tagged SP1 . The cells were fixed and sonicated, and DNA was immunoprecipitated with isotype control (IgG), V5, or STAT-1 antibodies. (Left) Relative enrichment of the Fbxl2 promoter was quantified by qPCR. (Right) Agarose gel of a 193-bp region of the Fbxl2 core promoter amplified by PCR. (H) C2C12 cells were transfected with plasmids expressing V5-tagged SP1 wild type (Wt), SP1-T739A, and SP1-T739D for 48 h. The cells were fixed and sonicated, and DNA was immunoprecipitated (IP) with V5 antibody. (Left) Relative enrichment of the Fbxl2 promoter was quantified by qPCR. (Right) Agarose gel of the 193-bp region of the Fbxl2 core promoter amplified by PCR. ns, not significant ( P > 0.05); *, P

Techniques Used: Activity Assay, Expressing, Transfection, Sonication, Immunoprecipitation, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification, Polymerase Chain Reaction

Identification and characterization of the proximal Fbxl2 promoter. (A) There was no significant difference in Fbxl2 mRNA degradation measured by qPCR at 3, 6, 9, and 12 h under control and TNF-α treatment conditions in an actinomycin D assay. (B) The half-life of Fbxl2 mRNA was estimated as 10.4 h (95% confidence interval [gray lines], 7.6 to 16.3) by linear regression analysis. (C) A 3,000-nucleotide (nt) region was cloned into the PGL3 basic firefly luciferase (Luc) reporter plasmid and then cotransfected into C2C12 cells with a nanoluciferase reporter as a transfection control. The cells were allowed to differentiate for 24 h prior to harvesting and lysis, and luminescence was measured using the NanoLuc dual-reporter assay (Promega). Sequential deletion of the reporter plasmid identified peak activity in the Fbxl2 promoter at nt −200 to +42 proximal to the TSS. (D) Additional deletion analysis of the reporter plasmid in the Fbxl2 promoter at nt −200 to +42 proximal to the TSS. Loss of constitutive reporter activity was identified between nt +160 and +120, with further loss of activity occurring with progressive deletion of the core promoter. Loss of TNF-α responsiveness also occurred between nt +160 and +120 of the TSS. (E) Schematic of a nt +240 to −42 insert within the PGL3 basic reporter construct showing three SP1 motifs proximal to the TSS. (F) Site-directed mutagenesis was performed to evaluate the impact of individual SP1 mutations on Fbxl2 core promoter activity; mutation in each SP1 site (X) resulted in a similar 25-fold loss of reporter activity, yet there was no additional loss of activity when all three SP1 sites were mutated. In the presence of TNF-α, each SP1 mutant plasmid did not show any additional loss of reporter activity, suggesting that SP1 is a TNF-α-responsive cis -acting element within the Fbxl2 promoter. The data are representative of the results of three independent experiments. ns, not significant ( P > 0.05); *, P
Figure Legend Snippet: Identification and characterization of the proximal Fbxl2 promoter. (A) There was no significant difference in Fbxl2 mRNA degradation measured by qPCR at 3, 6, 9, and 12 h under control and TNF-α treatment conditions in an actinomycin D assay. (B) The half-life of Fbxl2 mRNA was estimated as 10.4 h (95% confidence interval [gray lines], 7.6 to 16.3) by linear regression analysis. (C) A 3,000-nucleotide (nt) region was cloned into the PGL3 basic firefly luciferase (Luc) reporter plasmid and then cotransfected into C2C12 cells with a nanoluciferase reporter as a transfection control. The cells were allowed to differentiate for 24 h prior to harvesting and lysis, and luminescence was measured using the NanoLuc dual-reporter assay (Promega). Sequential deletion of the reporter plasmid identified peak activity in the Fbxl2 promoter at nt −200 to +42 proximal to the TSS. (D) Additional deletion analysis of the reporter plasmid in the Fbxl2 promoter at nt −200 to +42 proximal to the TSS. Loss of constitutive reporter activity was identified between nt +160 and +120, with further loss of activity occurring with progressive deletion of the core promoter. Loss of TNF-α responsiveness also occurred between nt +160 and +120 of the TSS. (E) Schematic of a nt +240 to −42 insert within the PGL3 basic reporter construct showing three SP1 motifs proximal to the TSS. (F) Site-directed mutagenesis was performed to evaluate the impact of individual SP1 mutations on Fbxl2 core promoter activity; mutation in each SP1 site (X) resulted in a similar 25-fold loss of reporter activity, yet there was no additional loss of activity when all three SP1 sites were mutated. In the presence of TNF-α, each SP1 mutant plasmid did not show any additional loss of reporter activity, suggesting that SP1 is a TNF-α-responsive cis -acting element within the Fbxl2 promoter. The data are representative of the results of three independent experiments. ns, not significant ( P > 0.05); *, P

Techniques Used: Real-time Polymerase Chain Reaction, Clone Assay, Luciferase, Plasmid Preparation, Transfection, Lysis, Reporter Assay, Activity Assay, Construct, Mutagenesis

12) Product Images from "Increased Binding of Specificity Protein 1 to the IL21R Promoter in B Cells Results in Enhanced B Cell Responses in Rheumatoid Arthritis"

Article Title: Increased Binding of Specificity Protein 1 to the IL21R Promoter in B Cells Results in Enhanced B Cell Responses in Rheumatoid Arthritis

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01978

Increased IL-21R expression correlates with increased SP1 binding to the IL-21R promoter in RA. (A) Total B cells were isolated from whole blood from RA patients and controls. SP1 binding to the IL21R promoter region and negative control IgG binding were determined using ChIP-qPCR analysis. The results are presented relative to input DNA. Significance was assessed using the student's t -test; n = 4 [Control (black circle), RA High (gray triangle)] n = 3 [RA Low (white square)]. ** p
Figure Legend Snippet: Increased IL-21R expression correlates with increased SP1 binding to the IL-21R promoter in RA. (A) Total B cells were isolated from whole blood from RA patients and controls. SP1 binding to the IL21R promoter region and negative control IgG binding were determined using ChIP-qPCR analysis. The results are presented relative to input DNA. Significance was assessed using the student's t -test; n = 4 [Control (black circle), RA High (gray triangle)] n = 3 [RA Low (white square)]. ** p

Techniques Used: Expressing, Binding Assay, Isolation, Negative Control, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Increased SP1 expression correlates with increased IL-21R expression in memory B cells in RA subjects (A) SP1 protein levels were assessed by flow cytometry in control ( n = 6, black circles), RA low IL-21R expressers ( n = 6; open squares) and RA high IL-21R expressers ( n = 5; gray triangles) in CD20 + and memory B cells (CD20 + CD38 − CD24 + ) (left). SP1 protein levels from left were correlated with IL-21R protein expression in total B cells in RA and control subjects ( n = 17) (right). (B) Representative histogram of mRNA levels from a no probe negative control, SP1 and positive control probe, RPL13A . (C) (left) SP1 mRNA levels were determined in total memory B cells in controls ( n = 7, black circles), RA low IL-21R expressers ( n = 7; open squares) and RA high IL-21R expressers ( n = 6; gray triangles). (right) SP1 mRNA levels from left were correlated with IL-21R protein expression in memory B cells in RA and control subjects combined ( n = 20). Significance was determined using Mann Whitney U tests (to compare RA-IL-21 high to controls and RA-IL-21 low ) and correlations were assessed with Pearson correlations.
Figure Legend Snippet: Increased SP1 expression correlates with increased IL-21R expression in memory B cells in RA subjects (A) SP1 protein levels were assessed by flow cytometry in control ( n = 6, black circles), RA low IL-21R expressers ( n = 6; open squares) and RA high IL-21R expressers ( n = 5; gray triangles) in CD20 + and memory B cells (CD20 + CD38 − CD24 + ) (left). SP1 protein levels from left were correlated with IL-21R protein expression in total B cells in RA and control subjects ( n = 17) (right). (B) Representative histogram of mRNA levels from a no probe negative control, SP1 and positive control probe, RPL13A . (C) (left) SP1 mRNA levels were determined in total memory B cells in controls ( n = 7, black circles), RA low IL-21R expressers ( n = 7; open squares) and RA high IL-21R expressers ( n = 6; gray triangles). (right) SP1 mRNA levels from left were correlated with IL-21R protein expression in memory B cells in RA and control subjects combined ( n = 20). Significance was determined using Mann Whitney U tests (to compare RA-IL-21 high to controls and RA-IL-21 low ) and correlations were assessed with Pearson correlations.

Techniques Used: Expressing, Flow Cytometry, Cytometry, Negative Control, Positive Control, MANN-WHITNEY

13) Product Images from "Angiotensin II AT2 receptor decreases AT1 receptor expression and function via nitric oxide/cGMP/Sp1 in renal proximal tubule cells from Wistar-Kyoto rats"

Article Title: Angiotensin II AT2 receptor decreases AT1 receptor expression and function via nitric oxide/cGMP/Sp1 in renal proximal tubule cells from Wistar-Kyoto rats

Journal: Journal of hypertension

doi: 10.1097/HJH.0b013e3283532099

Role of Sp1 in the inhibition of AT 1 receptor expression by AT 2 receptor stimulation in rat RPT cells. (a) Effect of Sp1 blocker in the inhibition of AT 1 receptor expression by AT 2 receptor stimulation in RPT cells. The RPT cells were incubated with the
Figure Legend Snippet: Role of Sp1 in the inhibition of AT 1 receptor expression by AT 2 receptor stimulation in rat RPT cells. (a) Effect of Sp1 blocker in the inhibition of AT 1 receptor expression by AT 2 receptor stimulation in RPT cells. The RPT cells were incubated with the

Techniques Used: Inhibition, Expressing, Incubation

14) Product Images from "Suppression of MicroRNA 200 Family Expression by Oncogenic KRAS Activation Promotes Cell Survival and Epithelial-Mesenchymal Transition in KRAS-Driven Cancer"

Article Title: Suppression of MicroRNA 200 Family Expression by Oncogenic KRAS Activation Promotes Cell Survival and Epithelial-Mesenchymal Transition in KRAS-Driven Cancer

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00079-16

KRAS suppresses mir-200 family expression through its downstream effectors JUN and SP1. (A) Downstream signaling pathways and effectors activated by KRAS. (B) Screening for effectors responsible for the suppressive function on mir-200 expression with
Figure Legend Snippet: KRAS suppresses mir-200 family expression through its downstream effectors JUN and SP1. (A) Downstream signaling pathways and effectors activated by KRAS. (B) Screening for effectors responsible for the suppressive function on mir-200 expression with

Techniques Used: Expressing

The signaling network connecting RAS and mir-200 regulates apoptosis and EMT in cancer cells. Through the signaling pathway RAF/MEK/ERK and transcription factors JUN and SP1, RAS suppresses mir-200 and hence stabilizes the expression level of BCL2 and
Figure Legend Snippet: The signaling network connecting RAS and mir-200 regulates apoptosis and EMT in cancer cells. Through the signaling pathway RAF/MEK/ERK and transcription factors JUN and SP1, RAS suppresses mir-200 and hence stabilizes the expression level of BCL2 and

Techniques Used: Expressing

15) Product Images from "Sp1 and Sp3 Are the Transcription Activators of Human ek1 Promoter in TSA-Treated Human Colon Carcinoma Cells"

Article Title: Sp1 and Sp3 Are the Transcription Activators of Human ek1 Promoter in TSA-Treated Human Colon Carcinoma Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0147886

ChIP analysis of ek1 minimal promoter region for the binding of Sp1 and Sp3 in HCT116 and HepG2 cells. PCR amplification products were resolved on 2% (w/v) agarose gel and visualized by EtBr staining. Band intensities were quantitated with Image J 1.42 and the relative intensities (compared to negative control) of PCR products from Sp1 and Sp3 immunoprecipitates were plotted. Each bar represents standard error of means (SEM) from two independent experiments. M: GeneRuler ™ DNA Ladder Mix; T: total input sample (unprocessed chromatin); P: positive control (amplified using GAPDH Primers) and N: pre-immune normal rabbit IgG (negative control).
Figure Legend Snippet: ChIP analysis of ek1 minimal promoter region for the binding of Sp1 and Sp3 in HCT116 and HepG2 cells. PCR amplification products were resolved on 2% (w/v) agarose gel and visualized by EtBr staining. Band intensities were quantitated with Image J 1.42 and the relative intensities (compared to negative control) of PCR products from Sp1 and Sp3 immunoprecipitates were plotted. Each bar represents standard error of means (SEM) from two independent experiments. M: GeneRuler ™ DNA Ladder Mix; T: total input sample (unprocessed chromatin); P: positive control (amplified using GAPDH Primers) and N: pre-immune normal rabbit IgG (negative control).

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Staining, Negative Control, Positive Control

Effects TSA treatment on the binding of Sp1, Sp3 and RNA polymerase II to the ek1 minimal promoter region. ChIP analysis was performed to confirm the interaction of (A) Sp proteins and (B) RNA polymerase II with the promoter under 1 μM TSA treatment for 24 hours. PCR amplification products were resolved on 2% (w/v) agarose gel and visualized by EtBr staining. Band intensities were quantitated with Image J 1.42 and the relative intensities (compared to negative control) of PCR products from Sp1 and Sp3 immunoprecipitates were plotted. Each bar represents standard error of means (SEM) from two independent experiments. M: GeneRuler ™ DNA Ladder Mix; T: total input sample (unprocessed chromatin); P: positive control (amplified using GAPDH primers) and N: pre-immune normal rabbit IgG (negative control).
Figure Legend Snippet: Effects TSA treatment on the binding of Sp1, Sp3 and RNA polymerase II to the ek1 minimal promoter region. ChIP analysis was performed to confirm the interaction of (A) Sp proteins and (B) RNA polymerase II with the promoter under 1 μM TSA treatment for 24 hours. PCR amplification products were resolved on 2% (w/v) agarose gel and visualized by EtBr staining. Band intensities were quantitated with Image J 1.42 and the relative intensities (compared to negative control) of PCR products from Sp1 and Sp3 immunoprecipitates were plotted. Each bar represents standard error of means (SEM) from two independent experiments. M: GeneRuler ™ DNA Ladder Mix; T: total input sample (unprocessed chromatin); P: positive control (amplified using GAPDH primers) and N: pre-immune normal rabbit IgG (negative control).

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Staining, Negative Control, Positive Control

16) Product Images from "HPV16 E6/E7 upregulate hTERC mRNA and gene amplification levels by relieving the effect of LKB1 on Sp1 phosphorylation in lung cancer cells"

Article Title: HPV16 E6/E7 upregulate hTERC mRNA and gene amplification levels by relieving the effect of LKB1 on Sp1 phosphorylation in lung cancer cells

Journal: Therapeutic Advances in Medical Oncology

doi: 10.1177/1758835920917562

The overexpression of E6 or E7 but low expression of LKB1, pSp1 (T739), and pSp1 (T453) were measured at 0, 12, 24, 36, and 48 hours in H1299 (a or b) and H460 (c or d) cells, and the protein expressions of E6, E7, LKB1, Sp1, pSp1 (T739), and pSp1 (T453) were detected by western blot in lung cancer cells. The low expression of Sp1 and hTERC was observed in A549 (e) and H1299 (f) cells, and the mRNA expression levels of both Sp1 and hTERC were quantified by qRT-PCR in lung cancer cells. The binding between Sp1 and the hTERC promoter region was shown by chromatin immunoprecipitation in H1299, H460, A549, and LK2 cells (g). NS, non-specific siRNA; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction; SiSp1, Sp1-specific siRNA. * p
Figure Legend Snippet: The overexpression of E6 or E7 but low expression of LKB1, pSp1 (T739), and pSp1 (T453) were measured at 0, 12, 24, 36, and 48 hours in H1299 (a or b) and H460 (c or d) cells, and the protein expressions of E6, E7, LKB1, Sp1, pSp1 (T739), and pSp1 (T453) were detected by western blot in lung cancer cells. The low expression of Sp1 and hTERC was observed in A549 (e) and H1299 (f) cells, and the mRNA expression levels of both Sp1 and hTERC were quantified by qRT-PCR in lung cancer cells. The binding between Sp1 and the hTERC promoter region was shown by chromatin immunoprecipitation in H1299, H460, A549, and LK2 cells (g). NS, non-specific siRNA; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction; SiSp1, Sp1-specific siRNA. * p

Techniques Used: Over Expression, Expressing, Western Blot, Quantitative RT-PCR, Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction

The overexpression of LKB1 but low expression of Sp1 and hTERC was observed in A549 (a) and LK2 (b) cells, whereas the low expression of LKB1 but overexpression of Sp1 and hTERC was observed in H1299 (c) and H460 (d) cells. Detection of both protein and mRNA expression of LKB1 and Sp1 by western blot and qRT-PCR, of the mRNA expression of hTERC by qRT-PCR, and of the transcriptional activity of Sp1 by a luciferase reporter assay was performed in lung cancer cells as well as in control cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Mock, mock transfection or mock-specific siRNA; NS, non-specific siRNA; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction; SiLKB1, LKB1-specific siRNA; Vector, empty vector. * p
Figure Legend Snippet: The overexpression of LKB1 but low expression of Sp1 and hTERC was observed in A549 (a) and LK2 (b) cells, whereas the low expression of LKB1 but overexpression of Sp1 and hTERC was observed in H1299 (c) and H460 (d) cells. Detection of both protein and mRNA expression of LKB1 and Sp1 by western blot and qRT-PCR, of the mRNA expression of hTERC by qRT-PCR, and of the transcriptional activity of Sp1 by a luciferase reporter assay was performed in lung cancer cells as well as in control cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Mock, mock transfection or mock-specific siRNA; NS, non-specific siRNA; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction; SiLKB1, LKB1-specific siRNA; Vector, empty vector. * p

Techniques Used: Over Expression, Expressing, Western Blot, Quantitative RT-PCR, Activity Assay, Luciferase, Reporter Assay, Transfection, Polymerase Chain Reaction, Plasmid Preparation

The overexpression of E6, E7, Sp1, and hTERC but low expression of LKB1 was observed in H1299 (a) and H460 (b) cells, whereas low expression of E6, E7, Sp1, and hTERC but overexpression of LKB1 was observed in A549 (c) and LK2 (d) cells. Detection of both protein and mRNA expression of E6, E7, LKB1, and Sp1 by western blot and qRT-PCR, of the mRNA expression of hTERC by qRT-PCR, and of the transcriptional activity of Sp1 by a luciferase reporter was performed in lung cancer cells as well as in control cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Mock, mock transfection or mock-specific siRNA; NS, non-specific siRNA; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction; SiE6, E6-specific siRNA; SiE7, E7-specific siRNA; Vector, empty vector. * p
Figure Legend Snippet: The overexpression of E6, E7, Sp1, and hTERC but low expression of LKB1 was observed in H1299 (a) and H460 (b) cells, whereas low expression of E6, E7, Sp1, and hTERC but overexpression of LKB1 was observed in A549 (c) and LK2 (d) cells. Detection of both protein and mRNA expression of E6, E7, LKB1, and Sp1 by western blot and qRT-PCR, of the mRNA expression of hTERC by qRT-PCR, and of the transcriptional activity of Sp1 by a luciferase reporter was performed in lung cancer cells as well as in control cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Mock, mock transfection or mock-specific siRNA; NS, non-specific siRNA; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction; SiE6, E6-specific siRNA; SiE7, E7-specific siRNA; Vector, empty vector. * p

Techniques Used: Over Expression, Expressing, Western Blot, Quantitative RT-PCR, Activity Assay, Luciferase, Transfection, Polymerase Chain Reaction, Plasmid Preparation

17) Product Images from "Increased Binding of Specificity Protein 1 to the IL21R Promoter in B Cells Results in Enhanced B Cell Responses in Rheumatoid Arthritis"

Article Title: Increased Binding of Specificity Protein 1 to the IL21R Promoter in B Cells Results in Enhanced B Cell Responses in Rheumatoid Arthritis

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01978

Increased IL-21R expression correlates with increased SP1 binding to the IL-21R promoter in RA. (A) Total B cells were isolated from whole blood from RA patients and controls. SP1 binding to the IL21R promoter region and negative control IgG binding were determined using ChIP-qPCR analysis. The results are presented relative to input DNA. Significance was assessed using the student's t -test; n = 4 [Control (black circle), RA High (gray triangle)] n = 3 [RA Low (white square)]. ** p
Figure Legend Snippet: Increased IL-21R expression correlates with increased SP1 binding to the IL-21R promoter in RA. (A) Total B cells were isolated from whole blood from RA patients and controls. SP1 binding to the IL21R promoter region and negative control IgG binding were determined using ChIP-qPCR analysis. The results are presented relative to input DNA. Significance was assessed using the student's t -test; n = 4 [Control (black circle), RA High (gray triangle)] n = 3 [RA Low (white square)]. ** p

Techniques Used: Expressing, Binding Assay, Isolation, Negative Control, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Increased SP1 expression correlates with increased IL-21R expression in memory B cells in RA subjects (A) SP1 protein levels were assessed by flow cytometry in control ( n = 6, black circles), RA low IL-21R expressers ( n = 6; open squares) and RA high IL-21R expressers ( n = 5; gray triangles) in CD20 + and memory B cells (CD20 + CD38 − CD24 + ) (left). SP1 protein levels from left were correlated with IL-21R protein expression in total B cells in RA and control subjects ( n = 17) (right). (B) Representative histogram of mRNA levels from a no probe negative control, SP1 and positive control probe, RPL13A . (C) (left) SP1 mRNA levels were determined in total memory B cells in controls ( n = 7, black circles), RA low IL-21R expressers ( n = 7; open squares) and RA high IL-21R expressers ( n = 6; gray triangles). (right) SP1 mRNA levels from left were correlated with IL-21R protein expression in memory B cells in RA and control subjects combined ( n = 20). Significance was determined using Mann Whitney U tests (to compare RA-IL-21 high to controls and RA-IL-21 low ) and correlations were assessed with Pearson correlations.
Figure Legend Snippet: Increased SP1 expression correlates with increased IL-21R expression in memory B cells in RA subjects (A) SP1 protein levels were assessed by flow cytometry in control ( n = 6, black circles), RA low IL-21R expressers ( n = 6; open squares) and RA high IL-21R expressers ( n = 5; gray triangles) in CD20 + and memory B cells (CD20 + CD38 − CD24 + ) (left). SP1 protein levels from left were correlated with IL-21R protein expression in total B cells in RA and control subjects ( n = 17) (right). (B) Representative histogram of mRNA levels from a no probe negative control, SP1 and positive control probe, RPL13A . (C) (left) SP1 mRNA levels were determined in total memory B cells in controls ( n = 7, black circles), RA low IL-21R expressers ( n = 7; open squares) and RA high IL-21R expressers ( n = 6; gray triangles). (right) SP1 mRNA levels from left were correlated with IL-21R protein expression in memory B cells in RA and control subjects combined ( n = 20). Significance was determined using Mann Whitney U tests (to compare RA-IL-21 high to controls and RA-IL-21 low ) and correlations were assessed with Pearson correlations.

Techniques Used: Expressing, Flow Cytometry, Cytometry, Negative Control, Positive Control, MANN-WHITNEY

18) Product Images from "BMP- specific SMADs function as novel repressors of PDGFA and modulate its expression in ovarian granulosa cells and tumors"

Article Title: BMP- specific SMADs function as novel repressors of PDGFA and modulate its expression in ovarian granulosa cells and tumors

Journal: Oncogene

doi: 10.1038/onc.2012.392

SMAD1/5 bind to the PDGFA promoter. (A) In silico promoter analysis identified several putative SMAD1/5 binding sites (gray shaded region) in the human PDGFA promoter region. Also shown are binding sites for Sp1 (dashed boxes), a known positive regulator for PDGFA promoter activity. The arrows in the promoter region indicate the sites where forward (-294F) and reverse (-82R) primers are designed for the ChIP assay. (B) Chromatin immunoprecipitation (ChIP) assay with an anti-SMAD1/5 antibody or control IgG was performed on cell extracts from COV434 cells demonstrated the in vivo binding of SMAD1/5 to the PDGFA promoter. (C) Real-time PCR analysis of immunoprecipitated DNA using locus specific primers demonstrated three-fold enrichment (ChIP/Input DNA) of SMAD1/5 occupancy at the PDGFA promoter compared to negative control (no locus-specific primers) and IgG. Asterisks indicate statistical significance (n=3; P
Figure Legend Snippet: SMAD1/5 bind to the PDGFA promoter. (A) In silico promoter analysis identified several putative SMAD1/5 binding sites (gray shaded region) in the human PDGFA promoter region. Also shown are binding sites for Sp1 (dashed boxes), a known positive regulator for PDGFA promoter activity. The arrows in the promoter region indicate the sites where forward (-294F) and reverse (-82R) primers are designed for the ChIP assay. (B) Chromatin immunoprecipitation (ChIP) assay with an anti-SMAD1/5 antibody or control IgG was performed on cell extracts from COV434 cells demonstrated the in vivo binding of SMAD1/5 to the PDGFA promoter. (C) Real-time PCR analysis of immunoprecipitated DNA using locus specific primers demonstrated three-fold enrichment (ChIP/Input DNA) of SMAD1/5 occupancy at the PDGFA promoter compared to negative control (no locus-specific primers) and IgG. Asterisks indicate statistical significance (n=3; P

Techniques Used: In Silico, Binding Assay, Activity Assay, Chromatin Immunoprecipitation, In Vivo, Real-time Polymerase Chain Reaction, Immunoprecipitation, Negative Control

Model of BR-Smad repression of PDGFA promoter activity. Earlier studies demonstrated that Sp1 mediates the basal transcription of the PDGFA gene through binding to four consensus binding sites in the proximal promoter region (denoted sites “A–D”). Based on ChIP data, we suggest that SMAD1 and SMAD5 bind to the proximal promoter region of PDGFA and block the “A” site for the Sp1 binding, thereby inhibiting gene expression. Loss of the BR-SMADs ( SMAD1 and SMAD5 ) in ovarian granulosa cells thus allows Sp1 to bind to the “A” site and leads to increased PDGFA activation, possibly initiated cell proliferation and contributing to disease onset.
Figure Legend Snippet: Model of BR-Smad repression of PDGFA promoter activity. Earlier studies demonstrated that Sp1 mediates the basal transcription of the PDGFA gene through binding to four consensus binding sites in the proximal promoter region (denoted sites “A–D”). Based on ChIP data, we suggest that SMAD1 and SMAD5 bind to the proximal promoter region of PDGFA and block the “A” site for the Sp1 binding, thereby inhibiting gene expression. Loss of the BR-SMADs ( SMAD1 and SMAD5 ) in ovarian granulosa cells thus allows Sp1 to bind to the “A” site and leads to increased PDGFA activation, possibly initiated cell proliferation and contributing to disease onset.

Techniques Used: Activity Assay, Binding Assay, Chromatin Immunoprecipitation, Blocking Assay, Expressing, Activation Assay

SMAD1/5 expression antagonizes Sp1 induction of PDGFA promoter. (A) COV434 cells were transiently co-transfected with 1 μg of -881:luc of the human PDGFA promoter and 0.5 μg of Sp1 and SMAD1/5 along with the renilla luciferase control plasmid (pRL-TK). Forty-eight hours after transfection, the cells were lysed and assessed for luciferase activity. Data are presented as the ratio of firefly luciferase to renilla. (B) Representative Western blot of whole cell lysates from COV434 cells transfected with either pcDNA3.1 or Flag-tagged SMAD1/5 expression plasmids and blotted with mouse anti-FLAG M2 antibody and mouse anti-β-actin antibody (loading control). (C) ChIP analysis in COV434 cells transfected with pcDNA3.1 or Flag-tagged SMAD1/5 expression plasmids. Chromatin cross-linked protein DNA complexes were immunoprecipitated with either anti-Sp1 antibody or with non-specific IgG and the PDGF-A promoter amplified by real-time PCR using locus specific primers. (D) SMAD1/5 represses the wild-type PDGFA promoter (-261:luc) but not the promoter bearing a mutation in the SMAD1/5 binding site (-261mutA: luc). COV434 cells were transiently co-transfected with 1 μg of -261:luc (control) or -261mutA: luc (mutant) and 0.5 μg of Sp1 and SMAD1/5 along with the renilla luciferase control plasmid (pRL-TK). Forty-eight hours after transfection, the cells were lysed and assessed for luciferase activity. Data are presented as the ratio of firefly luciferase to renilla. Different letters above the bars indicate statistically different means by ANOVA and post hoc analysis (n=4; P
Figure Legend Snippet: SMAD1/5 expression antagonizes Sp1 induction of PDGFA promoter. (A) COV434 cells were transiently co-transfected with 1 μg of -881:luc of the human PDGFA promoter and 0.5 μg of Sp1 and SMAD1/5 along with the renilla luciferase control plasmid (pRL-TK). Forty-eight hours after transfection, the cells were lysed and assessed for luciferase activity. Data are presented as the ratio of firefly luciferase to renilla. (B) Representative Western blot of whole cell lysates from COV434 cells transfected with either pcDNA3.1 or Flag-tagged SMAD1/5 expression plasmids and blotted with mouse anti-FLAG M2 antibody and mouse anti-β-actin antibody (loading control). (C) ChIP analysis in COV434 cells transfected with pcDNA3.1 or Flag-tagged SMAD1/5 expression plasmids. Chromatin cross-linked protein DNA complexes were immunoprecipitated with either anti-Sp1 antibody or with non-specific IgG and the PDGF-A promoter amplified by real-time PCR using locus specific primers. (D) SMAD1/5 represses the wild-type PDGFA promoter (-261:luc) but not the promoter bearing a mutation in the SMAD1/5 binding site (-261mutA: luc). COV434 cells were transiently co-transfected with 1 μg of -261:luc (control) or -261mutA: luc (mutant) and 0.5 μg of Sp1 and SMAD1/5 along with the renilla luciferase control plasmid (pRL-TK). Forty-eight hours after transfection, the cells were lysed and assessed for luciferase activity. Data are presented as the ratio of firefly luciferase to renilla. Different letters above the bars indicate statistically different means by ANOVA and post hoc analysis (n=4; P

Techniques Used: Expressing, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Western Blot, Chromatin Immunoprecipitation, Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction, Mutagenesis, Binding Assay

19) Product Images from "Effect of Tolfenamic Acid on Canine Cancer Cell Proliferation, Specificity Protein (Sp) Transcription Factors, and Sp-Regulated Proteins in Canine Osteosarcoma, Mammary Carcinoma, and Melanoma Cells"

Article Title: Effect of Tolfenamic Acid on Canine Cancer Cell Proliferation, Specificity Protein (Sp) Transcription Factors, and Sp-Regulated Proteins in Canine Osteosarcoma, Mammary Carcinoma, and Melanoma Cells

Journal: Journal of veterinary internal medicine / American College of Veterinary Internal Medicine

doi: 10.1111/j.1939-1676.2012.00931.x

Tolfenamic acid downregulates Sp1, Sp3, and Sp4 proteins in canine cancer cells. Canine cancer cell lines were treated with DMSO or 50 μM TA for 48 hours and whole cell lysates were analyzed for Sp1, Sp3, and Sp4 proteins by western blot analysis
Figure Legend Snippet: Tolfenamic acid downregulates Sp1, Sp3, and Sp4 proteins in canine cancer cells. Canine cancer cell lines were treated with DMSO or 50 μM TA for 48 hours and whole cell lysates were analyzed for Sp1, Sp3, and Sp4 proteins by western blot analysis

Techniques Used: Western Blot

Immunostaining of canine tumors and normal tissue for Sp1 protein. Fixed tumor/nontumor tissue from various archived tissue samples of 5 osteosarcomas, 5 melanomas, and 5 mammary carcinomas were stained for Sp1 protein as described in the Materials and
Figure Legend Snippet: Immunostaining of canine tumors and normal tissue for Sp1 protein. Fixed tumor/nontumor tissue from various archived tissue samples of 5 osteosarcomas, 5 melanomas, and 5 mammary carcinomas were stained for Sp1 protein as described in the Materials and

Techniques Used: Immunostaining, Staining

20) Product Images from "Protein Kinase C?-Induced Derepression of the Human Luteinizing Hormone Receptor Gene Transcription through ERK-Mediated Release of HDAC1/Sin3A Repressor Complex from Sp1 Sites"

Article Title: Protein Kinase C?-Induced Derepression of the Human Luteinizing Hormone Receptor Gene Transcription through ERK-Mediated Release of HDAC1/Sin3A Repressor Complex from Sp1 Sites

Journal: Molecular Endocrinology

doi: 10.1210/me.2008-0035

Requirement of Both Sp1/Sp3 Binding Sites and Involvement of Sp1 But Not Sp3 in PMA-Mediated hLHR Promoter Activation
Figure Legend Snippet: Requirement of Both Sp1/Sp3 Binding Sites and Involvement of Sp1 But Not Sp3 in PMA-Mediated hLHR Promoter Activation

Techniques Used: Binding Assay, Activation Assay

PMA Treatement Enhances Sp1 Phosphorylation in a PKCα-Dependent Manner
Figure Legend Snippet: PMA Treatement Enhances Sp1 Phosphorylation in a PKCα-Dependent Manner

Techniques Used:

ERK Interacts with Sp1 and Is Recruited to the hLHR Promoter in the Presence of PMA
Figure Legend Snippet: ERK Interacts with Sp1 and Is Recruited to the hLHR Promoter in the Presence of PMA

Techniques Used:

Related Articles

other:

Article Title: Tumor Necrosis Factor Alpha Regulates Skeletal Myogenesis by Inhibiting SP1 Interaction with cis-Acting Regulatory Elements within the Fbxl2 Gene Promoter
Article Snippet: TRAF6 antibody (catalog no. 06-1110) and SP1 antibody with isotype control antibody (catalog no. 17-601) were purchased from Millipore (Cambridge, MA). p38-MAPK (catalog no. 9212) and phosphorylated p38-MAPK (p-p38-MAPK) (catalog no. 4511), Akt (catalog no. 2920S), p-Akt-Ser473 (catalog no. 4060S), p-Akt-Thr308 (catalog no. 13038S), ERK1/2 (catalog no. 9101S), and p-ERK1/2 (catalog no. 4696S) were obtained from Cell Signaling (Beverly, MA).

Incubation:

Article Title: Inhibition of Notch1 signaling overcomes resistance to the death ligand Trail by specificity protein 1-dependent upregulation of death receptor 5
Article Snippet: .. After clearance of non-specifically bound chromatin fragments, supernatants equivalent to 25 μ g DNA were incubated with 2 μ g of Sp1 antibody or 2 μ g of rabbit IgG antibody as control (both provided in the same kit; Millipore, Billerica, MA, USA, #17-601; Lot: JBC1920619). ..

Immunoprecipitation:

Article Title: Regulation of HK2 expression through alterations in CpG methylation of the HK2 promoter during progression of hepatocellular carcinoma
Article Snippet: .. Chromatin samples were immunoprecipitated with anti-H3 (ab1791; Abcam, Cambridge, UK), anti-H3K4me3 (ab8580; Abcam), anti-H3K9me3 (ab8898; Abcam), anti-H3K27me3 (ab6002; Abcam), anti-HIF-1α, anti-Sp1 (EMD Millipore, Temecula, CA, USA) and control IgG (sc-2027; Santa Cruz) at 4°C overnight, and then proteins were harvested with mouse anti-IgG Ab linked to magnetic protein A & G beads (Dynal, Lake Success, NY, USA). ..

Binding Assay:

Article Title: Distinct chromatin structures at the monoamine oxidase‐A promoter correlate with allele‐specific expression in SH‐SY5Y cells, et al. Distinct chromatin structures at the monoamine oxidase‐A promoter correlate with allele‐specific expression in SH‐SY5Y cells
Article Snippet: .. The immunoprecipitations were performed using the following antibodies: anti‐Histone H3 (Abcam, Cambridge, UK), anti‐Nucleolin (Abcam), anti‐hnRNP K (Abcam), anti‐CCHC‐Type Zinc Finger Nucleic Acid Binding Protein gene (CNBP) (Abcam), anti‐RNA pol II CTD phospho Ser5 (Active Motif, Carlsbad, California), anti‐Sp1 and anti‐CCCTC‐Binding Factor gene (CTCF) (Millipore, Nottingham, UK). ..

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  • 97
    Millipore sp1 antibody
    Increased IL-21R expression correlates with increased <t>SP1</t> binding to the IL-21R promoter in RA. (A) Total B cells were isolated from whole blood from RA patients and controls. SP1 binding to the IL21R promoter region and negative control IgG binding were determined using ChIP-qPCR analysis. The results are presented relative to input DNA. Significance was assessed using the student's t -test; n = 4 [Control (black circle), RA High (gray triangle)] n = 3 [RA Low (white square)]. ** p
    Sp1 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore sp1
    G9a regulates DKK1 expression through <t>Sp1/p300</t> in a methyltransferase activity dependent manner. (A) Sp1 occupancy was analysed by ChIP-PCR at the DKK1 promoter. IgG was used as a control (n=2). Values correspond to the average ± SD. ( B-D ) Sp1, p300 and H3K9ac enrichment at the DKK1 promoter was analysed in 2.5 μM of UNC0642 treated cells compared to DMSO controls. The dot plots show reduced enrichment in UNC0642 treated cells (n=3). Values correspond to the average ± SEM. ( E ) PLA was done to examine Sp1 and p300 interaction in control and 2.5 μM of UNC0642 treated cells. Images were captured using confocal microscopy. The dot plot shows the number of dots per nuclei in UNC0642 treated cells compared to control cells (n=3). Each dot represents an interaction. Values correspond to the average ± SEM. ( F-G ) qPCR analysis of CDK1 mRNA in control and siG9a RD18 cells and in control and 2.5 μM of UNC0642 treated RD18 cells (n=3). Values correspond to the average ± SEM. ( H-I ) CDK1 protein was analysed in in control and siG9a RD18 cells and in DMSO and 2.5 μM of UNC0642 treated RD18 cells. Representative images from 2 independent experiments are shown. ( J ) ChIP-seq analysis in RD18 cells showed G9a occupancy at different regions of the chromatin. ( K ) Snapshot of G9a binding peak at the CDK1 promoter from the UCSC genome browser. ( L ) G9a occupancy at the CDK1 promoter was validated by ChIP-PCR (n=3). The dot plot shows enrichment compared to IgG which was used as a control. Values correspond to the average ± SEM. Statistical significance in A, B, C, D, E, F, G and L was calculated by unpaired two-tailed t test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.
    Sp1, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti sp1 antibody
    Ras overexpression increases association of RbAp46 with HDAC1 at the <t>SP1</t> site of RECK promoter in 7–4 cells. (A) The 7–4 cells were transiently transfected with plasmid DNA pcDNA-RbAp46 (0.2 μg) or RbAp46-specific siRNA (55.6 nM) in the presence of IPTG for 48 hr. After treatment, co-immunoprecipitation was conducted using anti-RbAp46, anti-HDAC1 antibodies, or anti-Sp1 antibody. (B) The cells were co-transfected with 0.2 μg of plasmid DNA of pGL3-RECK and pGL3-Sp1 mutant in the presence or absence of pcDNA-RbAp46 plasmid. RECK promoter activity was measured at 48 hr post-transfection. **: statistical significance at p
    Anti Sp1 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore anti sp1 rabbit polyclonal igg
    Transcription factors <t>Sp1</t> is involved in AMACR gene regulation in HCT 116 cells. Putative Sp1 binding site at CG3 and 10 were identified. A: ChIP assay with Sp1 antibody targeting AMACR CGI ( Figure 2B ). A PCR signal was detected in the Sp1 antibody ChIP with genomic DNA and normal <t>IgG-immunoprecipitated</t> DNA as the PCR input and negative control, respectively ( Figure 5A, top panel ). As a ChIP negative control, amplification of a region in the last exon of AMACR gene distant to the putative Sp1 sites was included in the experiment. Only the DNA input showed the amplification ( Figure 5A, lower panel ). B: siRNA-mediated Sp1 knockdown decreased the AMACR transcript level. Real-time RT-PCR demonstrated that the first-round siSp1 decreased the Sp1 transcript level 48% ( p
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    Increased IL-21R expression correlates with increased SP1 binding to the IL-21R promoter in RA. (A) Total B cells were isolated from whole blood from RA patients and controls. SP1 binding to the IL21R promoter region and negative control IgG binding were determined using ChIP-qPCR analysis. The results are presented relative to input DNA. Significance was assessed using the student's t -test; n = 4 [Control (black circle), RA High (gray triangle)] n = 3 [RA Low (white square)]. ** p

    Journal: Frontiers in Immunology

    Article Title: Increased Binding of Specificity Protein 1 to the IL21R Promoter in B Cells Results in Enhanced B Cell Responses in Rheumatoid Arthritis

    doi: 10.3389/fimmu.2018.01978

    Figure Lengend Snippet: Increased IL-21R expression correlates with increased SP1 binding to the IL-21R promoter in RA. (A) Total B cells were isolated from whole blood from RA patients and controls. SP1 binding to the IL21R promoter region and negative control IgG binding were determined using ChIP-qPCR analysis. The results are presented relative to input DNA. Significance was assessed using the student's t -test; n = 4 [Control (black circle), RA High (gray triangle)] n = 3 [RA Low (white square)]. ** p

    Article Snippet: Conditions for sonication: 20% duty cycle, 30 min. Sonicated samples were immunoprecipitated with magnetically labeled SP1 antibody or control IgG overnight at 4°C.

    Techniques: Expressing, Binding Assay, Isolation, Negative Control, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Increased SP1 expression correlates with increased IL-21R expression in memory B cells in RA subjects (A) SP1 protein levels were assessed by flow cytometry in control ( n = 6, black circles), RA low IL-21R expressers ( n = 6; open squares) and RA high IL-21R expressers ( n = 5; gray triangles) in CD20 + and memory B cells (CD20 + CD38 − CD24 + ) (left). SP1 protein levels from left were correlated with IL-21R protein expression in total B cells in RA and control subjects ( n = 17) (right). (B) Representative histogram of mRNA levels from a no probe negative control, SP1 and positive control probe, RPL13A . (C) (left) SP1 mRNA levels were determined in total memory B cells in controls ( n = 7, black circles), RA low IL-21R expressers ( n = 7; open squares) and RA high IL-21R expressers ( n = 6; gray triangles). (right) SP1 mRNA levels from left were correlated with IL-21R protein expression in memory B cells in RA and control subjects combined ( n = 20). Significance was determined using Mann Whitney U tests (to compare RA-IL-21 high to controls and RA-IL-21 low ) and correlations were assessed with Pearson correlations.

    Journal: Frontiers in Immunology

    Article Title: Increased Binding of Specificity Protein 1 to the IL21R Promoter in B Cells Results in Enhanced B Cell Responses in Rheumatoid Arthritis

    doi: 10.3389/fimmu.2018.01978

    Figure Lengend Snippet: Increased SP1 expression correlates with increased IL-21R expression in memory B cells in RA subjects (A) SP1 protein levels were assessed by flow cytometry in control ( n = 6, black circles), RA low IL-21R expressers ( n = 6; open squares) and RA high IL-21R expressers ( n = 5; gray triangles) in CD20 + and memory B cells (CD20 + CD38 − CD24 + ) (left). SP1 protein levels from left were correlated with IL-21R protein expression in total B cells in RA and control subjects ( n = 17) (right). (B) Representative histogram of mRNA levels from a no probe negative control, SP1 and positive control probe, RPL13A . (C) (left) SP1 mRNA levels were determined in total memory B cells in controls ( n = 7, black circles), RA low IL-21R expressers ( n = 7; open squares) and RA high IL-21R expressers ( n = 6; gray triangles). (right) SP1 mRNA levels from left were correlated with IL-21R protein expression in memory B cells in RA and control subjects combined ( n = 20). Significance was determined using Mann Whitney U tests (to compare RA-IL-21 high to controls and RA-IL-21 low ) and correlations were assessed with Pearson correlations.

    Article Snippet: Conditions for sonication: 20% duty cycle, 30 min. Sonicated samples were immunoprecipitated with magnetically labeled SP1 antibody or control IgG overnight at 4°C.

    Techniques: Expressing, Flow Cytometry, Cytometry, Negative Control, Positive Control, MANN-WHITNEY

    G9a regulates DKK1 expression through Sp1/p300 in a methyltransferase activity dependent manner. (A) Sp1 occupancy was analysed by ChIP-PCR at the DKK1 promoter. IgG was used as a control (n=2). Values correspond to the average ± SD. ( B-D ) Sp1, p300 and H3K9ac enrichment at the DKK1 promoter was analysed in 2.5 μM of UNC0642 treated cells compared to DMSO controls. The dot plots show reduced enrichment in UNC0642 treated cells (n=3). Values correspond to the average ± SEM. ( E ) PLA was done to examine Sp1 and p300 interaction in control and 2.5 μM of UNC0642 treated cells. Images were captured using confocal microscopy. The dot plot shows the number of dots per nuclei in UNC0642 treated cells compared to control cells (n=3). Each dot represents an interaction. Values correspond to the average ± SEM. ( F-G ) qPCR analysis of CDK1 mRNA in control and siG9a RD18 cells and in control and 2.5 μM of UNC0642 treated RD18 cells (n=3). Values correspond to the average ± SEM. ( H-I ) CDK1 protein was analysed in in control and siG9a RD18 cells and in DMSO and 2.5 μM of UNC0642 treated RD18 cells. Representative images from 2 independent experiments are shown. ( J ) ChIP-seq analysis in RD18 cells showed G9a occupancy at different regions of the chromatin. ( K ) Snapshot of G9a binding peak at the CDK1 promoter from the UCSC genome browser. ( L ) G9a occupancy at the CDK1 promoter was validated by ChIP-PCR (n=3). The dot plot shows enrichment compared to IgG which was used as a control. Values correspond to the average ± SEM. Statistical significance in A, B, C, D, E, F, G and L was calculated by unpaired two-tailed t test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

    Journal: bioRxiv

    Article Title: Deregulation of the histone H3K9 di-methylation landscape suppresses canonical Wnt signaling in embryonal rhabdomyosarcoma

    doi: 10.1101/2020.04.20.050120

    Figure Lengend Snippet: G9a regulates DKK1 expression through Sp1/p300 in a methyltransferase activity dependent manner. (A) Sp1 occupancy was analysed by ChIP-PCR at the DKK1 promoter. IgG was used as a control (n=2). Values correspond to the average ± SD. ( B-D ) Sp1, p300 and H3K9ac enrichment at the DKK1 promoter was analysed in 2.5 μM of UNC0642 treated cells compared to DMSO controls. The dot plots show reduced enrichment in UNC0642 treated cells (n=3). Values correspond to the average ± SEM. ( E ) PLA was done to examine Sp1 and p300 interaction in control and 2.5 μM of UNC0642 treated cells. Images were captured using confocal microscopy. The dot plot shows the number of dots per nuclei in UNC0642 treated cells compared to control cells (n=3). Each dot represents an interaction. Values correspond to the average ± SEM. ( F-G ) qPCR analysis of CDK1 mRNA in control and siG9a RD18 cells and in control and 2.5 μM of UNC0642 treated RD18 cells (n=3). Values correspond to the average ± SEM. ( H-I ) CDK1 protein was analysed in in control and siG9a RD18 cells and in DMSO and 2.5 μM of UNC0642 treated RD18 cells. Representative images from 2 independent experiments are shown. ( J ) ChIP-seq analysis in RD18 cells showed G9a occupancy at different regions of the chromatin. ( K ) Snapshot of G9a binding peak at the CDK1 promoter from the UCSC genome browser. ( L ) G9a occupancy at the CDK1 promoter was validated by ChIP-PCR (n=3). The dot plot shows enrichment compared to IgG which was used as a control. Values correspond to the average ± SEM. Statistical significance in A, B, C, D, E, F, G and L was calculated by unpaired two-tailed t test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

    Article Snippet: The following antibodies were used for ChIP assays: ChIP-grade anti-G9a (Abcam), anti-H3K9ac (Abcam), Sp1 (Rabbit Millipore) and p300 (Abcam).

    Techniques: Expressing, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Proximity Ligation Assay, Confocal Microscopy, Real-time Polymerase Chain Reaction, Binding Assay, Two Tailed Test

    G9a regulates Sp1 and p300 occupancy at the DKK1 promoter. (A-B) ChIP-PCR analysis showed decrease in p300 occupancy and H3K9ac enrichment at the DKK1 promoter in shG9a RD cells as compared to control (n=2). Error bars indicate the mean ± SD. (C) PLA was used to determine G9a-Sp1 interaction which did not change upon UNC0642 treatment. Data shown is representative of 2 independent experiments. Error bars indicate the mean ± SD. (D) G9a-p300 interaction was not significantly altered by UNC0642 treatment. Data shown is representative of 2 independent experiments. Error bars indicate the mean ± SD. Statistical significance in A, B, C and D was calculated as unpaired two-tailed t test. ** P ≤ 0.001, ns = not significant. (E, F) CDK1 mRNA was analysed in control and siG9a RD cells and in control and UNC0642 treated RD cells (n=3). Values correspond to the average ± SEM. (H, I) CDK1 protein were analysed in in control and siG9a RD18 cells and in DMSO and UNC0642 treated RD18 cells. The image is representative of 2 independent experiments. Statistical significance in E and F was calculated by unpaired two-tailed t test. *** P ≤ 0.001 and **** P ≤ 0.0001.

    Journal: bioRxiv

    Article Title: Deregulation of the histone H3K9 di-methylation landscape suppresses canonical Wnt signaling in embryonal rhabdomyosarcoma

    doi: 10.1101/2020.04.20.050120

    Figure Lengend Snippet: G9a regulates Sp1 and p300 occupancy at the DKK1 promoter. (A-B) ChIP-PCR analysis showed decrease in p300 occupancy and H3K9ac enrichment at the DKK1 promoter in shG9a RD cells as compared to control (n=2). Error bars indicate the mean ± SD. (C) PLA was used to determine G9a-Sp1 interaction which did not change upon UNC0642 treatment. Data shown is representative of 2 independent experiments. Error bars indicate the mean ± SD. (D) G9a-p300 interaction was not significantly altered by UNC0642 treatment. Data shown is representative of 2 independent experiments. Error bars indicate the mean ± SD. Statistical significance in A, B, C and D was calculated as unpaired two-tailed t test. ** P ≤ 0.001, ns = not significant. (E, F) CDK1 mRNA was analysed in control and siG9a RD cells and in control and UNC0642 treated RD cells (n=3). Values correspond to the average ± SEM. (H, I) CDK1 protein were analysed in in control and siG9a RD18 cells and in DMSO and UNC0642 treated RD18 cells. The image is representative of 2 independent experiments. Statistical significance in E and F was calculated by unpaired two-tailed t test. *** P ≤ 0.001 and **** P ≤ 0.0001.

    Article Snippet: The following antibodies were used for ChIP assays: ChIP-grade anti-G9a (Abcam), anti-H3K9ac (Abcam), Sp1 (Rabbit Millipore) and p300 (Abcam).

    Techniques: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Proximity Ligation Assay, Two Tailed Test

    Ras overexpression increases association of RbAp46 with HDAC1 at the SP1 site of RECK promoter in 7–4 cells. (A) The 7–4 cells were transiently transfected with plasmid DNA pcDNA-RbAp46 (0.2 μg) or RbAp46-specific siRNA (55.6 nM) in the presence of IPTG for 48 hr. After treatment, co-immunoprecipitation was conducted using anti-RbAp46, anti-HDAC1 antibodies, or anti-Sp1 antibody. (B) The cells were co-transfected with 0.2 μg of plasmid DNA of pGL3-RECK and pGL3-Sp1 mutant in the presence or absence of pcDNA-RbAp46 plasmid. RECK promoter activity was measured at 48 hr post-transfection. **: statistical significance at p

    Journal: BMC Cancer

    Article Title: Ras induces experimental lung metastasis through up-regulation of RbAp46 to suppress RECK promoter activity

    doi: 10.1186/s12885-015-1155-7

    Figure Lengend Snippet: Ras overexpression increases association of RbAp46 with HDAC1 at the SP1 site of RECK promoter in 7–4 cells. (A) The 7–4 cells were transiently transfected with plasmid DNA pcDNA-RbAp46 (0.2 μg) or RbAp46-specific siRNA (55.6 nM) in the presence of IPTG for 48 hr. After treatment, co-immunoprecipitation was conducted using anti-RbAp46, anti-HDAC1 antibodies, or anti-Sp1 antibody. (B) The cells were co-transfected with 0.2 μg of plasmid DNA of pGL3-RECK and pGL3-Sp1 mutant in the presence or absence of pcDNA-RbAp46 plasmid. RECK promoter activity was measured at 48 hr post-transfection. **: statistical significance at p

    Article Snippet: After sonication, the resulting soluble chromatin was diluted 1:10 with ChIP dilution buffer and immunoprecipitated by anti-Sp1 antibody (Millipore, Billerica, MA, USA), anti-RbAp46 antibody (Abcam, Cambridge, MA, USA) or control IgG.

    Techniques: Over Expression, Transfection, Plasmid Preparation, Immunoprecipitation, Mutagenesis, Activity Assay

    A schematic hypothetical model shows that RbAp46 is a Ras up-regulated gene which participates in Ras-induced experimental lung metastasis through binding with HDAC1 and Sp1 to suppress RECK expression followed by MMP-9 activation and metastasis.

    Journal: BMC Cancer

    Article Title: Ras induces experimental lung metastasis through up-regulation of RbAp46 to suppress RECK promoter activity

    doi: 10.1186/s12885-015-1155-7

    Figure Lengend Snippet: A schematic hypothetical model shows that RbAp46 is a Ras up-regulated gene which participates in Ras-induced experimental lung metastasis through binding with HDAC1 and Sp1 to suppress RECK expression followed by MMP-9 activation and metastasis.

    Article Snippet: After sonication, the resulting soluble chromatin was diluted 1:10 with ChIP dilution buffer and immunoprecipitated by anti-Sp1 antibody (Millipore, Billerica, MA, USA), anti-RbAp46 antibody (Abcam, Cambridge, MA, USA) or control IgG.

    Techniques: Binding Assay, Expressing, Activation Assay

    Transcription factors Sp1 is involved in AMACR gene regulation in HCT 116 cells. Putative Sp1 binding site at CG3 and 10 were identified. A: ChIP assay with Sp1 antibody targeting AMACR CGI ( Figure 2B ). A PCR signal was detected in the Sp1 antibody ChIP with genomic DNA and normal IgG-immunoprecipitated DNA as the PCR input and negative control, respectively ( Figure 5A, top panel ). As a ChIP negative control, amplification of a region in the last exon of AMACR gene distant to the putative Sp1 sites was included in the experiment. Only the DNA input showed the amplification ( Figure 5A, lower panel ). B: siRNA-mediated Sp1 knockdown decreased the AMACR transcript level. Real-time RT-PCR demonstrated that the first-round siSp1 decreased the Sp1 transcript level 48% ( p

    Journal: PLoS Genetics

    Article Title: Deletion Hotspots in AMACR Promoter CpG Island Are cis-Regulatory Elements Controlling the Gene Expression in the Colon

    doi: 10.1371/journal.pgen.1000334

    Figure Lengend Snippet: Transcription factors Sp1 is involved in AMACR gene regulation in HCT 116 cells. Putative Sp1 binding site at CG3 and 10 were identified. A: ChIP assay with Sp1 antibody targeting AMACR CGI ( Figure 2B ). A PCR signal was detected in the Sp1 antibody ChIP with genomic DNA and normal IgG-immunoprecipitated DNA as the PCR input and negative control, respectively ( Figure 5A, top panel ). As a ChIP negative control, amplification of a region in the last exon of AMACR gene distant to the putative Sp1 sites was included in the experiment. Only the DNA input showed the amplification ( Figure 5A, lower panel ). B: siRNA-mediated Sp1 knockdown decreased the AMACR transcript level. Real-time RT-PCR demonstrated that the first-round siSp1 decreased the Sp1 transcript level 48% ( p

    Article Snippet: A total of 7.5 µg of anti-Sp1 rabbit polyclonal IgG (cat. no. 07-645, Upstate/Millipore) was used in each IP.

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Immunoprecipitation, Negative Control, Amplification, Quantitative RT-PCR