Structured Review

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
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1) Product Images from "Ras induces experimental lung metastasis through up-regulation of RbAp46 to suppress RECK promoter activity"

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

Journal: BMC Cancer

doi: 10.1186/s12885-015-1155-7

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
Figure Legend 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

Techniques Used: 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.
Figure Legend 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.

Techniques Used: Binding Assay, Expressing, Activation Assay

2) Product Images from "Sp1 and Sp3 transcription factors regulate the basal expression of human microsomal epoxide hydrolase (EPHX1) through interaction with the proximal E1b far upstream promoter"

Article Title: Sp1 and Sp3 transcription factors regulate the basal expression of human microsomal epoxide hydrolase (EPHX1) through interaction with the proximal E1b far upstream promoter

Journal: Gene

doi: 10.1016/j.gene.2013.11.053

ChIP assay for Sp1 and Sp3 binding to the E1b proximal promoter in BEAS-2B and C3A cells
Figure Legend Snippet: ChIP assay for Sp1 and Sp3 binding to the E1b proximal promoter in BEAS-2B and C3A cells

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

Identification and mutational analysis of Sp1/Sp3 binding sites within the E1b proximal promoter region
Figure Legend Snippet: Identification and mutational analysis of Sp1/Sp3 binding sites within the E1b proximal promoter region

Techniques Used: Binding Assay

Sp1 and Sp3 regulated E1b proximal promoter activities
Figure Legend Snippet: Sp1 and Sp3 regulated E1b proximal promoter activities

Techniques Used:

EMSA and supershift analyses show Sp1 binding to putative Sp1/Sp3 binding sites on E1b-300 promoter
Figure Legend Snippet: EMSA and supershift analyses show Sp1 binding to putative Sp1/Sp3 binding sites on E1b-300 promoter

Techniques Used: Binding Assay

3) Product Images from "PI3K activation increases SDF-1 production and number of osteoclast precursors, and enhances SDF-1-mediated osteoclast precursor migration"

Article Title: PI3K activation increases SDF-1 production and number of osteoclast precursors, and enhances SDF-1-mediated osteoclast precursor migration

Journal: Bone Reports

doi: 10.1016/j.bonr.2019.100203

Proposed model depicting the role of PI3K/AKT/Sp1 axis on SDF-1 expression in CAR cells and the effect of increased SDF-1 levels on osteoclast precursor migration in response to increased PI3K activation. Loss of Cbl-PI3K interaction results in increased PI3K activity, which leads to increased phosphorylation of AKT. Sp1, an important substrate of PI3K is activated, translocated to the nucleus and binds to the SDF-1 promoter regions to activate SDF-1 transcription in CAR cells. Sp1 binding to SDF-1 promoter regions is inhibited by Mithramycin treatment resulting in decreased SDF-1 transcription to a lesser extent in YF cells compared to wild type cells due to increased Sp1 activation in YF cells. Increased SDF-1 gene expression leads to increased SDF-1 protein levels, which stimulate migration of osteoclast precursors expressing SDF-1 receptor, CXCR4. AMD3100 blocks CXCR4 activation by SDF-1 and prevents osteoclast precursor migration, to a lesser extent in YF cells compared to wild type cells. Increased osteoclast precursor migration might lead to increased recruitment to the bone marrow milieu finally contributing to increased number of osteoclasts in YF mice lacking Cbl-PI3K interaction.
Figure Legend Snippet: Proposed model depicting the role of PI3K/AKT/Sp1 axis on SDF-1 expression in CAR cells and the effect of increased SDF-1 levels on osteoclast precursor migration in response to increased PI3K activation. Loss of Cbl-PI3K interaction results in increased PI3K activity, which leads to increased phosphorylation of AKT. Sp1, an important substrate of PI3K is activated, translocated to the nucleus and binds to the SDF-1 promoter regions to activate SDF-1 transcription in CAR cells. Sp1 binding to SDF-1 promoter regions is inhibited by Mithramycin treatment resulting in decreased SDF-1 transcription to a lesser extent in YF cells compared to wild type cells due to increased Sp1 activation in YF cells. Increased SDF-1 gene expression leads to increased SDF-1 protein levels, which stimulate migration of osteoclast precursors expressing SDF-1 receptor, CXCR4. AMD3100 blocks CXCR4 activation by SDF-1 and prevents osteoclast precursor migration, to a lesser extent in YF cells compared to wild type cells. Increased osteoclast precursor migration might lead to increased recruitment to the bone marrow milieu finally contributing to increased number of osteoclasts in YF mice lacking Cbl-PI3K interaction.

Techniques Used: Expressing, Migration, Activation Assay, Activity Assay, Binding Assay, Mouse Assay

Sp1 mediated transcription of SDF-1. A. Nuclear protein from cells treated with LY294002 for 3 h at different concentrations was subjected to Western blotting. Blot was probed with antibody for SP1. The blot was stripped and reprobed for actin (in cytoplasm) and tubulin (in nucleus) to show the relative absence of cytoplasmic proteins in the nuclear lysate. Sp1/tubulin ratio is shown below the Western blot. B. Cells were treated with Mithramycin (inhibits Sp1 binding to GC-rich DNA) at indicated concentrations for 3 h and SDF-1 mRNA levels were determined by qRT-PCR analysis. Values are shown as relative to untreated WT cells. * A representative of 3 independent experiments is shown and P value of
Figure Legend Snippet: Sp1 mediated transcription of SDF-1. A. Nuclear protein from cells treated with LY294002 for 3 h at different concentrations was subjected to Western blotting. Blot was probed with antibody for SP1. The blot was stripped and reprobed for actin (in cytoplasm) and tubulin (in nucleus) to show the relative absence of cytoplasmic proteins in the nuclear lysate. Sp1/tubulin ratio is shown below the Western blot. B. Cells were treated with Mithramycin (inhibits Sp1 binding to GC-rich DNA) at indicated concentrations for 3 h and SDF-1 mRNA levels were determined by qRT-PCR analysis. Values are shown as relative to untreated WT cells. * A representative of 3 independent experiments is shown and P value of

Techniques Used: Western Blot, Binding Assay, Quantitative RT-PCR

4) Product Images from "miR-7a/b attenuates post-myocardial infarction remodeling and protects H9c2 cardiomyoblast against hypoxia-induced apoptosis involving Sp1 and PARP-1"

Article Title: miR-7a/b attenuates post-myocardial infarction remodeling and protects H9c2 cardiomyoblast against hypoxia-induced apoptosis involving Sp1 and PARP-1

Journal: Scientific Reports

doi: 10.1038/srep29082

miR-7a/b repressed the downstream target Sp1 expression in vivo and in vitro . ( A ) Western blots of Sp1 in cells exposed to hypoxia for different time, ( B,C ) Western blots showing the effect of miR-7a/b on regulation of Sp1 in vitro ( B ) and in vivo ( C ). ( D ) Immunostaining of Sp1 in vivo (scale bar: 20 μm). ( E ) Conserved miR-7a/b binding sites and mutated binding sites in 3′ untranslated region (UTR) of Sp1. ( F ) Luciferase activity analysis. Con: sham mice without LAD occlusion. MI: mice with LAD occlusion. Con: normal cultured H9c2 cells ( A,B ) or sham mice without LAD occlusion ( C,D ). MI: mice with LAD occlusion. NC: H9c2 cells exposed to hypoxia for 12 h. Data are the mean ± SD, n = 3/group ( A–C , F ), n = 6/group ( D ), *p
Figure Legend Snippet: miR-7a/b repressed the downstream target Sp1 expression in vivo and in vitro . ( A ) Western blots of Sp1 in cells exposed to hypoxia for different time, ( B,C ) Western blots showing the effect of miR-7a/b on regulation of Sp1 in vitro ( B ) and in vivo ( C ). ( D ) Immunostaining of Sp1 in vivo (scale bar: 20 μm). ( E ) Conserved miR-7a/b binding sites and mutated binding sites in 3′ untranslated region (UTR) of Sp1. ( F ) Luciferase activity analysis. Con: sham mice without LAD occlusion. MI: mice with LAD occlusion. Con: normal cultured H9c2 cells ( A,B ) or sham mice without LAD occlusion ( C,D ). MI: mice with LAD occlusion. NC: H9c2 cells exposed to hypoxia for 12 h. Data are the mean ± SD, n = 3/group ( A–C , F ), n = 6/group ( D ), *p

Techniques Used: Expressing, In Vivo, In Vitro, Western Blot, Immunostaining, Binding Assay, Luciferase, Activity Assay, Mouse Assay, Cell Culture

Sp1 binding activity mediated miR-7a/b-regulated Sp1, PARP-1 and caspase-3 expression in hypoxia H9c2 cells. ( A,D–F ) Western blots of Sp1, PARP-1 and caspase-3 in hypoxic cells pretreated with different concentration of mithramycin (nM). ( C,G–I ) Western blots of Sp1, PARP-1 and caspase-3 in hypoxic H9c2 cells transfected with miR-7a/b inhibitors that treated with or without 100 nM mithramycin. ( B ) Representative ChIP assays. Con: normal cultured H9c2 cells, M: mithramycin, *p
Figure Legend Snippet: Sp1 binding activity mediated miR-7a/b-regulated Sp1, PARP-1 and caspase-3 expression in hypoxia H9c2 cells. ( A,D–F ) Western blots of Sp1, PARP-1 and caspase-3 in hypoxic cells pretreated with different concentration of mithramycin (nM). ( C,G–I ) Western blots of Sp1, PARP-1 and caspase-3 in hypoxic H9c2 cells transfected with miR-7a/b inhibitors that treated with or without 100 nM mithramycin. ( B ) Representative ChIP assays. Con: normal cultured H9c2 cells, M: mithramycin, *p

Techniques Used: Binding Assay, Activity Assay, Expressing, Western Blot, Concentration Assay, Transfection, Chromatin Immunoprecipitation, Cell Culture

5) Product Images from "SP1 induced long non-coding RNA LINC00958 overexpression facilitate cell proliferation, migration and invasion in lung adenocarcinoma via mediating miR-625-5p/CPSF7 axis"

Article Title: SP1 induced long non-coding RNA LINC00958 overexpression facilitate cell proliferation, migration and invasion in lung adenocarcinoma via mediating miR-625-5p/CPSF7 axis

Journal: Cancer Cell International

doi: 10.1186/s12935-020-1099-0

SP1 induces LINC00958 overexpression by serving as a transcription activator. a QRT-PCR analysis was performed to investigate the expression of SP1 after transfecting with pcDNA3.1 and pcDNA3.1/SP1. b QRT-PCR was performed to study the effect of pcDNA3.1/SP1 on LINC00958 expression. c The putative SP1 binding motif in − 2000 bp of human LINC00958 promoter. d LINC00958 promoter was divided into four sectional fragments. e ChIP assay using antibody targeting SP1 and IgG was performed to detect the affinity of SP1 with LINC00958 promoter. f The full LINC00958 promoter (P FL) and LINC00958 P1 deleted (P D) was constructed. g ChIP assay using antibody targeting SP1 and IgG was performed to determine the relative enrichment of LINC00958 promoter. h pGL3-P1-WT, pGL3-P1-MUT1 and pGL3-P1-MUT2 luciferase vectors were constructed. i HEK-293T cells were transfected with pcDNA3.1 and pcDNA3.1/SP1 and luciferase reporter assays was performed among P1-WT, P1-MUT1 and P1-MUT2. **P
Figure Legend Snippet: SP1 induces LINC00958 overexpression by serving as a transcription activator. a QRT-PCR analysis was performed to investigate the expression of SP1 after transfecting with pcDNA3.1 and pcDNA3.1/SP1. b QRT-PCR was performed to study the effect of pcDNA3.1/SP1 on LINC00958 expression. c The putative SP1 binding motif in − 2000 bp of human LINC00958 promoter. d LINC00958 promoter was divided into four sectional fragments. e ChIP assay using antibody targeting SP1 and IgG was performed to detect the affinity of SP1 with LINC00958 promoter. f The full LINC00958 promoter (P FL) and LINC00958 P1 deleted (P D) was constructed. g ChIP assay using antibody targeting SP1 and IgG was performed to determine the relative enrichment of LINC00958 promoter. h pGL3-P1-WT, pGL3-P1-MUT1 and pGL3-P1-MUT2 luciferase vectors were constructed. i HEK-293T cells were transfected with pcDNA3.1 and pcDNA3.1/SP1 and luciferase reporter assays was performed among P1-WT, P1-MUT1 and P1-MUT2. **P

Techniques Used: Over Expression, Quantitative RT-PCR, Expressing, Binding Assay, Chromatin Immunoprecipitation, Construct, Luciferase, Transfection

6) Product Images from "Krüppel –Like Factor 8 is a Stress-Responsive Transcription Factor that Regulates Expression of HuR"

Article Title: Krüppel –Like Factor 8 is a Stress-Responsive Transcription Factor that Regulates Expression of HuR

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

doi: 10.1159/000363019

A putative Sp/KLF binding region just upstream of the transcriptional start site binds a factor that is not Sp1. (A) Schematic of the proximal promoter region of HuR. (B) Sequences of the sense strand of probes used in protein-DNA binding assays. Underlined
Figure Legend Snippet: A putative Sp/KLF binding region just upstream of the transcriptional start site binds a factor that is not Sp1. (A) Schematic of the proximal promoter region of HuR. (B) Sequences of the sense strand of probes used in protein-DNA binding assays. Underlined

Techniques Used: Binding Assay

7) Product Images from "Ultraviolet-B induces ERCC6 repression in lens epithelium cells of age-related nuclear cataract through coordinated DNA hypermethylation and histone deacetylation"

Article Title: Ultraviolet-B induces ERCC6 repression in lens epithelium cells of age-related nuclear cataract through coordinated DNA hypermethylation and histone deacetylation

Journal: Clinical Epigenetics

doi: 10.1186/s13148-016-0229-y

UVB irradiation decreased the occupancy of endogenous Sp1 and H3K9 acetylation on the ERCC6 promoter, but increased occupancy of DNMT3b and HDAC1 on the ERCC6 promoter. a HLE-B3 cells were treated with UVB irradiation. ChIP was performed using anti-Sp1, anti-DNMT3b, anti-HDAC1, and anti-ac-H3 (K9). b Statistically significant differences from control cells were indicated by *( P
Figure Legend Snippet: UVB irradiation decreased the occupancy of endogenous Sp1 and H3K9 acetylation on the ERCC6 promoter, but increased occupancy of DNMT3b and HDAC1 on the ERCC6 promoter. a HLE-B3 cells were treated with UVB irradiation. ChIP was performed using anti-Sp1, anti-DNMT3b, anti-HDAC1, and anti-ac-H3 (K9). b Statistically significant differences from control cells were indicated by *( P

Techniques Used: Irradiation, Chromatin Immunoprecipitation

Knockdown of endogenous Sp1 decreased the mRNA expression and protein level of ERCC6 and Sp1 in 239T cells. a Detection of ERCC6 and Sp1 mRNA expression by qRT-PCR. Values represent mean ± SD. * P
Figure Legend Snippet: Knockdown of endogenous Sp1 decreased the mRNA expression and protein level of ERCC6 and Sp1 in 239T cells. a Detection of ERCC6 and Sp1 mRNA expression by qRT-PCR. Values represent mean ± SD. * P

Techniques Used: Expressing, Quantitative RT-PCR

Electrophoretic mobility shift detected the DNA-binding ability of Sp1 on the ERCC6 promoter. The DNA-protein complex ( indicated by black arrow ) can be observed ( lanes 3 , 4 , 5 , 7 , and 9 ). In contrast, no nucleoprotein complex was observed when used the M-probe and labeled mutated type probe ( lanes 1 and 8 ). The competition assay was done by the addition of 50-, 100-, or 200-fold molar excess of unlabeled methylated probes to the incubation mixtures (lanes 4, 5, and 6). The complex formation was fully suppressed by the addition of a 200-fold molar excess of unlabeled wild type probe (lane 6) and not was suppressed by 200-fold molar excess of unlabeled mutated type probe (lane 9). A supershift band (indicated by black arrowhead , lane 7) was detected when anti-Sp1 antibody was incubated
Figure Legend Snippet: Electrophoretic mobility shift detected the DNA-binding ability of Sp1 on the ERCC6 promoter. The DNA-protein complex ( indicated by black arrow ) can be observed ( lanes 3 , 4 , 5 , 7 , and 9 ). In contrast, no nucleoprotein complex was observed when used the M-probe and labeled mutated type probe ( lanes 1 and 8 ). The competition assay was done by the addition of 50-, 100-, or 200-fold molar excess of unlabeled methylated probes to the incubation mixtures (lanes 4, 5, and 6). The complex formation was fully suppressed by the addition of a 200-fold molar excess of unlabeled wild type probe (lane 6) and not was suppressed by 200-fold molar excess of unlabeled mutated type probe (lane 9). A supershift band (indicated by black arrowhead , lane 7) was detected when anti-Sp1 antibody was incubated

Techniques Used: Electrophoretic Mobility Shift Assay, Binding Assay, Labeling, Competitive Binding Assay, Methylation, Incubation

Methylation status of the CpG island at promoter of ERCC6 in LECs of controls and ARNCs (30 controls and 30 ARNCs, the samples cover those analyzed in Fig. 1 ). a The CpG island (-603/-396, black arrow ) located in the promoter of ERCC6 . There are twelve CpG sites ( the red marker letter ) and a putative binding site for Sp1 (-446/-437) relative to transcriptional starting site ( red arrow ) in the region. b In LECs of ARNCs, the CpG site 8 displayed hypermethylation compared to the controls. * P
Figure Legend Snippet: Methylation status of the CpG island at promoter of ERCC6 in LECs of controls and ARNCs (30 controls and 30 ARNCs, the samples cover those analyzed in Fig. 1 ). a The CpG island (-603/-396, black arrow ) located in the promoter of ERCC6 . There are twelve CpG sites ( the red marker letter ) and a putative binding site for Sp1 (-446/-437) relative to transcriptional starting site ( red arrow ) in the region. b In LECs of ARNCs, the CpG site 8 displayed hypermethylation compared to the controls. * P

Techniques Used: Methylation, Marker, Binding Assay

8) Product Images from "DDX3X Biomarker Correlates with Poor Survival in Human Gliomas"

Article Title: DDX3X Biomarker Correlates with Poor Survival in Human Gliomas

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms160715578

The protein–protein interaction (PPI) network and ingenuity pathway analysis (IPA). ( A ) The PPI network, as shown in the confidence view, created by the STRING database. DDX3X is controlling hub, and black squares show the protein clusters; ( B ) The IPA predicted signaling pathways transcriptionally regulated through DDX3X; and ( C ) The DDX3X-regulated genes including ESR1, and transcription factor SP1 were validated in normal brain tissue and human glioma cell lines. Protein lysates of glioma cell lines including LN229, U87MG, GBM8401 and U118MG were applied to SDS-PAGE and Western blot analysis to quantitate DDX3X, ESR1, and SP1 protein expression. These results are representative of two independent experiments. β-actin served as a loading control.
Figure Legend Snippet: The protein–protein interaction (PPI) network and ingenuity pathway analysis (IPA). ( A ) The PPI network, as shown in the confidence view, created by the STRING database. DDX3X is controlling hub, and black squares show the protein clusters; ( B ) The IPA predicted signaling pathways transcriptionally regulated through DDX3X; and ( C ) The DDX3X-regulated genes including ESR1, and transcription factor SP1 were validated in normal brain tissue and human glioma cell lines. Protein lysates of glioma cell lines including LN229, U87MG, GBM8401 and U118MG were applied to SDS-PAGE and Western blot analysis to quantitate DDX3X, ESR1, and SP1 protein expression. These results are representative of two independent experiments. β-actin served as a loading control.

Techniques Used: Indirect Immunoperoxidase Assay, SDS Page, Western Blot, Expressing

9) Product Images from "Cigarette Smoke Induces MUC5AC Protein Expression through the Activation of Sp1 *"

Article Title: Cigarette Smoke Induces MUC5AC Protein Expression through the Activation of Sp1 *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.334375

Sp1 cis -elements mediate CSE-induced MUC5AC gene promoter transactivation in A549 cells. A , relative luciferase activity in cells transfected with a 3×Sp1 tandem repeat promoter-firefly luciferase reporter gene with and without 3% CSE treatment.
Figure Legend Snippet: Sp1 cis -elements mediate CSE-induced MUC5AC gene promoter transactivation in A549 cells. A , relative luciferase activity in cells transfected with a 3×Sp1 tandem repeat promoter-firefly luciferase reporter gene with and without 3% CSE treatment.

Techniques Used: Luciferase, Activity Assay, Transfection

Expression and nuclear localization of Sp1 is induced by CSE. A , A549 cells grown in submerged cell culture conditions were treated with or without 3% CSE for 4 h followed by harvest of cytoplasmic and nuclear protein for Western blots using anti-Sp1
Figure Legend Snippet: Expression and nuclear localization of Sp1 is induced by CSE. A , A549 cells grown in submerged cell culture conditions were treated with or without 3% CSE for 4 h followed by harvest of cytoplasmic and nuclear protein for Western blots using anti-Sp1

Techniques Used: Expressing, Cell Culture, Western Blot

Two Sp1 cis -elements are required to mediate CSE-induced MUC5AC gene promoter trans-activation in A549 cells. A , schematic representation of the four luciferase reporter vectors driven by the CSE-responsive −3752/−3337 bp promoter region
Figure Legend Snippet: Two Sp1 cis -elements are required to mediate CSE-induced MUC5AC gene promoter trans-activation in A549 cells. A , schematic representation of the four luciferase reporter vectors driven by the CSE-responsive −3752/−3337 bp promoter region

Techniques Used: Activation Assay, Luciferase

Pharmacological Sp1 inhibitor mithramycin A blocked CSE-induced MUC5AC promoter luciferase activity. A , relative luciferase activity in cells transfected with a luciferase reporter vector driven by a 3.7-kb MUC5AC promoter (MUC5AC) with or without 3%
Figure Legend Snippet: Pharmacological Sp1 inhibitor mithramycin A blocked CSE-induced MUC5AC promoter luciferase activity. A , relative luciferase activity in cells transfected with a luciferase reporter vector driven by a 3.7-kb MUC5AC promoter (MUC5AC) with or without 3%

Techniques Used: Luciferase, Activity Assay, Transfection, Plasmid Preparation

Identification and characterization of two CSE-induced Sp1-DNA binding sites on the MUC5AC promoter. A549 cells were grown to confluence followed by treatment with or without 3% CSE exposure for 2 h followed by harvest of nuclear protein for EMSA analyses.
Figure Legend Snippet: Identification and characterization of two CSE-induced Sp1-DNA binding sites on the MUC5AC promoter. A549 cells were grown to confluence followed by treatment with or without 3% CSE exposure for 2 h followed by harvest of nuclear protein for EMSA analyses.

Techniques Used: Binding Assay

10) Product Images from "Western blot data using two distinct anti-O-GlcNAc monoclonal antibodies showing unique glycosylation status on cellular proteins under 2-deoxy-d-glucose treatment"

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

Journal: Data in Brief

doi: 10.1016/j.dib.2016.12.001

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

Techniques Used: Western Blot

11) Product Images from "Hypoxia induces downregulation of soluble guanylyl cyclase ?1 by miR-34c-5p"

Article Title: Hypoxia induces downregulation of soluble guanylyl cyclase ?1 by miR-34c-5p

Journal: Journal of Cell Science

doi: 10.1242/jcs.113381

Summary diagram of the hypoxia–Sp1–miR-34c-5p signaling that regulates sGCβ 1 subunit expression. Hypoxia upregulates Sp1 expression. Sp1 in turn transcriptionally promotes miR-34c-5p expression by direct interaction with the promoter
Figure Legend Snippet: Summary diagram of the hypoxia–Sp1–miR-34c-5p signaling that regulates sGCβ 1 subunit expression. Hypoxia upregulates Sp1 expression. Sp1 in turn transcriptionally promotes miR-34c-5p expression by direct interaction with the promoter

Techniques Used: Expressing

Sp1 transcriptionally regulates miR-34c-5p expression during hypoxia. ( A ) mRNA expression of Sp1 in mouse lungs exposed to hypoxia for 72 hours or 120 hours. Data are expressed as means ± s.e.m ( n = 3; *P
Figure Legend Snippet: Sp1 transcriptionally regulates miR-34c-5p expression during hypoxia. ( A ) mRNA expression of Sp1 in mouse lungs exposed to hypoxia for 72 hours or 120 hours. Data are expressed as means ± s.e.m ( n = 3; *P

Techniques Used: Expressing

12) Product Images from "Evidence that TSC2 acts as a transcription factor and binds to and represses the promoter of Epiregulin"

Article Title: Evidence that TSC2 acts as a transcription factor and binds to and represses the promoter of Epiregulin

Journal: Nucleic Acids Research

doi: 10.1093/nar/gku278

ChIP analysis to determine in vivo binding of TSC2 to the EREG promoter. ( A ) Diagrammatic representation of the sub-regions 1 and 2 of the promoter along with the primer pairs. ( B ) TSC2 binds to the promoter region from −857 bp to −303 bp. The INP lane represents the amplification from sheared chromatin as a template, and NT is no template control. ( C ) The promoter region from −325 bp to +165 bp was negative for TSC2 binding. ( D ) The relative quantity of promoter enriched by ChIP was quantified by qPCR and expressed as the fold enrichment of the promoter sub-regions 1 and 2 after normalization to a rabbit IgG. UT represents untransfected cells. TSC2 siRNA was used at a final concentration of 100 nM. Data represent the means of two independent experiments. ( E ) DNA binding of SP1 transcription factor to the P1 promoter of the SLC22A18AS gene as a surrogate positive control for the ChIP protocol. Note, the amplification in the +Ab lane with primers SP1F and SP1R.
Figure Legend Snippet: ChIP analysis to determine in vivo binding of TSC2 to the EREG promoter. ( A ) Diagrammatic representation of the sub-regions 1 and 2 of the promoter along with the primer pairs. ( B ) TSC2 binds to the promoter region from −857 bp to −303 bp. The INP lane represents the amplification from sheared chromatin as a template, and NT is no template control. ( C ) The promoter region from −325 bp to +165 bp was negative for TSC2 binding. ( D ) The relative quantity of promoter enriched by ChIP was quantified by qPCR and expressed as the fold enrichment of the promoter sub-regions 1 and 2 after normalization to a rabbit IgG. UT represents untransfected cells. TSC2 siRNA was used at a final concentration of 100 nM. Data represent the means of two independent experiments. ( E ) DNA binding of SP1 transcription factor to the P1 promoter of the SLC22A18AS gene as a surrogate positive control for the ChIP protocol. Note, the amplification in the +Ab lane with primers SP1F and SP1R.

Techniques Used: Chromatin Immunoprecipitation, In Vivo, Binding Assay, Amplification, Real-time Polymerase Chain Reaction, Concentration Assay, Positive Control

13) Product Images from "Cystathionine β-synthase regulates endothelial function via protein S-sulfhydration"

Article Title: Cystathionine β-synthase regulates endothelial function via protein S-sulfhydration

Journal: The FASEB Journal

doi: 10.1096/fj.15-278648

A , B ) Charge-reduced, isotope-deconvoluted MSE spectra (fragment ion matched profiles) of untreated and 100 µM NaHS-treated full-length Sp1 protein. *The site of sulfhydration. C ) ChIP assay to determine relative Sp1 binding to the VEGFR-2 promoter in scrambled or CBS siRNA- or CBS siRNA + NaHS (2 mM)-treated HUVECs.
Figure Legend Snippet: A , B ) Charge-reduced, isotope-deconvoluted MSE spectra (fragment ion matched profiles) of untreated and 100 µM NaHS-treated full-length Sp1 protein. *The site of sulfhydration. C ) ChIP assay to determine relative Sp1 binding to the VEGFR-2 promoter in scrambled or CBS siRNA- or CBS siRNA + NaHS (2 mM)-treated HUVECs.

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

Related Articles

Incubation:

Article Title: Sp1 and Sp3 transcription factors regulate the basal expression of human microsomal epoxide hydrolase (EPHX1) through interaction with the proximal E1b far upstream promoter
Article Snippet: The diluted chromatin was pre-cleared overnight at 4°C with 35 µL protein A/G Plus-agarose beads (sc-2003, Santa Cruz Biotechnology) which were pre-blocked with sonicated salmon sperm DNA (201190, Stratagene, Santa Clara, CA) and BSA (2930, EM Science, Billerica, MA). .. Pre-cleaned chromatin was then incubated overnight at 4°C with 4 µg of anti-Sp1 antibody (17-601, Millipore), anti-Sp1 antibody (sc-14027, Santa Cruz Biotechnology), anti-Sp3 antibody (sc-644, Santa Cruz Biotechnology) or normal rabbit IgG (2729s, Cell Signaling Technology). .. To collect the antibody-chromatin complex, 75 µL protein A/G Plus-agarose beads pre-blocked as above were added, incubated for 3 h with rotation at 4°C and pelleted by centrifugation at 5000×g for 1 min.

Article Title: Histopathologic and Immunohistochemical Studies of Keratoglobus
Article Snippet: Biotinylated donkey anti–goat IgG (1:250), donkey anti–rabbit IgG (1:250), and donkey anti–mouse IgG (1:250; all from Jackson ImmunoResearch, West Grove, Pennsylvania) were used as secondary antibodies at room temperature for 45 minutes. .. The color reaction for the anti-Sp1 was carried out with Fast Red TR/Naphthol AS-MX phosphate (Sigma, St Louis, Missouri) after sections were incubated with alkaline phosphatase–conjugated ExtrAvidin (1:50; Sigma). .. For α1-PI, MMP-1, MMP-2, and MMP-3, sections were incubated with avidin-biotin complex (Vectastain; Vector Laboratories, Burlingame, California) for 45 minutes followed by incubation with 3,3 diaminobenzidine tetrahydrochloride (Sigma).

Western Blot:

Article Title: PI3K activation increases SDF-1 production and number of osteoclast precursors, and enhances SDF-1-mediated osteoclast precursor migration
Article Snippet: Transwell chambers for migration assay were purchased from Corning (Tewksbury, MA). .. For western blotting analysis, anti-phospho-AKT (Thr308), anti-AKT, anti-phospho GSKα/β Ser21/9, anti-GSK-beta, anti-Actin, anti-Tubulin antibodies were purchased from Cell Signaling Technology (Danvers, MA) and anti-Sp1 antibody, IgG secondary antibody from Millipore (Temecula, CA). .. HBSS, HEPES and fetal calf serum were purchased from Gibco, Invitrogen (Carlsbad, CA).

Article Title: Human rpL3 induces G?/S arrest or apoptosis by modulating p21waf1/cip1 levels in a p53-independent manner
Article Snippet: .. Western blotting assay was performed as described previously, using antibodies anti-rpL3 (Primm), anti-FLAG (Sigma-Aldrich), anti-Sp1 (Millipore), anti-p21, anti-NPM, anti-HA, anti-His, anti-PARP and anti-α-tubulin (Santa Cruz Biotechnology). .. Chromatin immunoprecipitation (ChIP) assay was performed as previously reported, using anti-rpL3, anti-NPM, anti-Sp1 antibodies or normal mouse IgG.

Immunostaining:

Article Title: Specificity protein 1-zinc finger protein 179 pathway is involved in the attenuation of oxidative stress following brain injury
Article Snippet: 2.11 Immunofluorescence The cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) in PBS according to a method described previously . .. Immunostaining was conducted with anti-Sp1 (Millipore) and anti-Flag M2 (Sigma-Aldrich) antibodies. .. The cells were then treated with Alexa Fluor 488-conjugated goat anti-rabbit IgG and Alexa Fluor 568-conjugated goat anti-mouse IgG polyclonal antibodies (Invitrogen).

Sonication:

Article Title: Ras induces experimental lung metastasis through up-regulation of RbAp46 to suppress RECK promoter activity
Article Snippet: Chromatin immunoprecipitation (ChIP) assay The cells (2×106 cells/10 cm plate) were treated with IPTG (5 mM, Invitrogen, Boston, MA, USA) for 24 hr, and ChIP assay was conducted as previously described [ ]. .. 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. .. The chromatin-antibody complexes were incubated with salmon sperm DNA/Protein A Agarose-50% (Millipore Corp., Billerica, MA, USA) overnight at 4°C with rotation.

Chromatin Immunoprecipitation:

Article Title: Ras induces experimental lung metastasis through up-regulation of RbAp46 to suppress RECK promoter activity
Article Snippet: Chromatin immunoprecipitation (ChIP) assay The cells (2×106 cells/10 cm plate) were treated with IPTG (5 mM, Invitrogen, Boston, MA, USA) for 24 hr, and ChIP assay was conducted as previously described [ ]. .. 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. .. The chromatin-antibody complexes were incubated with salmon sperm DNA/Protein A Agarose-50% (Millipore Corp., Billerica, MA, USA) overnight at 4°C with rotation.

Immunoprecipitation:

Article Title: Ras induces experimental lung metastasis through up-regulation of RbAp46 to suppress RECK promoter activity
Article Snippet: Chromatin immunoprecipitation (ChIP) assay The cells (2×106 cells/10 cm plate) were treated with IPTG (5 mM, Invitrogen, Boston, MA, USA) for 24 hr, and ChIP assay was conducted as previously described [ ]. .. 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. .. The chromatin-antibody complexes were incubated with salmon sperm DNA/Protein A Agarose-50% (Millipore Corp., Billerica, MA, USA) overnight at 4°C with rotation.

Article Title: Proinflammatory Stimuli Control N-Acylphosphatidylethanolamine-Specific Phospholipase D Expression in Macrophages
Article Snippet: .. Immunoprecipitation was carried out with 5 μg of anti-Sp1 (Millipore Corporation, Billerica, MA) or anti-acetylhistone H3 (Millipore Corporation) antibodies at 4°C overnight with rotation. .. After immunoprecipitation, 20 μl of a mixture of salmon sperm DNA/protein A/protein G was added and incubated at 4°C with rotation for 3 h and followed by brief centrifugation.

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  • 93
    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
    https://www.bioz.com/result/anti sp1 antibody/product/Millipore
    Average 93 stars, based on 1 article reviews
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    anti sp1 antibody - by Bioz Stars, 2021-07
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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
    Anti Sp1 Rabbit Polyclonal Igg, 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
    https://www.bioz.com/result/anti sp1 rabbit polyclonal igg/product/Millipore
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti sp1 rabbit polyclonal igg - by Bioz Stars, 2021-07
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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
    https://www.bioz.com/result/sp1 antibody/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sp1 antibody - by Bioz Stars, 2021-07
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    N/A
    Specific protein 1 Sp1 is a transcription regulator that has a transactivation domain at the N terminus and a DNA binding domain at the C terminus that contain three zinc
      Buy from Supplier

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    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

    ChIP assay for Sp1 and Sp3 binding to the E1b proximal promoter in BEAS-2B and C3A cells

    Journal: Gene

    Article Title: Sp1 and Sp3 transcription factors regulate the basal expression of human microsomal epoxide hydrolase (EPHX1) through interaction with the proximal E1b far upstream promoter

    doi: 10.1016/j.gene.2013.11.053

    Figure Lengend Snippet: ChIP assay for Sp1 and Sp3 binding to the E1b proximal promoter in BEAS-2B and C3A cells

    Article Snippet: Pre-cleaned chromatin was then incubated overnight at 4°C with 4 µg of anti-Sp1 antibody (17-601, Millipore), anti-Sp1 antibody (sc-14027, Santa Cruz Biotechnology), anti-Sp3 antibody (sc-644, Santa Cruz Biotechnology) or normal rabbit IgG (2729s, Cell Signaling Technology).

    Techniques: Chromatin Immunoprecipitation, Binding Assay

    Identification and mutational analysis of Sp1/Sp3 binding sites within the E1b proximal promoter region

    Journal: Gene

    Article Title: Sp1 and Sp3 transcription factors regulate the basal expression of human microsomal epoxide hydrolase (EPHX1) through interaction with the proximal E1b far upstream promoter

    doi: 10.1016/j.gene.2013.11.053

    Figure Lengend Snippet: Identification and mutational analysis of Sp1/Sp3 binding sites within the E1b proximal promoter region

    Article Snippet: Pre-cleaned chromatin was then incubated overnight at 4°C with 4 µg of anti-Sp1 antibody (17-601, Millipore), anti-Sp1 antibody (sc-14027, Santa Cruz Biotechnology), anti-Sp3 antibody (sc-644, Santa Cruz Biotechnology) or normal rabbit IgG (2729s, Cell Signaling Technology).

    Techniques: Binding Assay

    Sp1 and Sp3 regulated E1b proximal promoter activities

    Journal: Gene

    Article Title: Sp1 and Sp3 transcription factors regulate the basal expression of human microsomal epoxide hydrolase (EPHX1) through interaction with the proximal E1b far upstream promoter

    doi: 10.1016/j.gene.2013.11.053

    Figure Lengend Snippet: Sp1 and Sp3 regulated E1b proximal promoter activities

    Article Snippet: Pre-cleaned chromatin was then incubated overnight at 4°C with 4 µg of anti-Sp1 antibody (17-601, Millipore), anti-Sp1 antibody (sc-14027, Santa Cruz Biotechnology), anti-Sp3 antibody (sc-644, Santa Cruz Biotechnology) or normal rabbit IgG (2729s, Cell Signaling Technology).

    Techniques:

    EMSA and supershift analyses show Sp1 binding to putative Sp1/Sp3 binding sites on E1b-300 promoter

    Journal: Gene

    Article Title: Sp1 and Sp3 transcription factors regulate the basal expression of human microsomal epoxide hydrolase (EPHX1) through interaction with the proximal E1b far upstream promoter

    doi: 10.1016/j.gene.2013.11.053

    Figure Lengend Snippet: EMSA and supershift analyses show Sp1 binding to putative Sp1/Sp3 binding sites on E1b-300 promoter

    Article Snippet: Pre-cleaned chromatin was then incubated overnight at 4°C with 4 µg of anti-Sp1 antibody (17-601, Millipore), anti-Sp1 antibody (sc-14027, Santa Cruz Biotechnology), anti-Sp3 antibody (sc-644, Santa Cruz Biotechnology) or normal rabbit IgG (2729s, Cell Signaling Technology).

    Techniques: Binding 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

    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