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Sp1 antibody

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The SP1 antibody is a laboratory reagent used in various research and diagnostic applications. It is a monoclonal antibody that specifically recognizes and binds to the SP1 transcription factor, which is a protein involved in the regulation of gene expression. The SP1 antibody can be used for techniques such as Western blotting, immunoprecipitation, and immunohistochemistry to detect and study the SP1 protein in biological samples.

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Lab products found in correlation

Hematoxylin, by Vector Laboratories (2 mentions) Dulbecco s modified eagle s medium, by Merck Group (2 mentions) β actin antibody, by Merck Group (2 mentions) Human b cell isolation kit 2, by Miltenyi Biotec (1 mentions) Truchip chromatin shearing reagent kit, by Covaris (1 mentions) Afa microtube, by Covaris (1 mentions) Qiaquick pcr purification kit, by Qiagen (1 mentions) M220 focused ultrasonicator, by Covaris (1 mentions) Pierce agarose chip kit, by Thermo Fisher Scientific (1 mentions) Integrin β1, by Santa Cruz Biotechnology (1 mentions) Formaldehyde, by Merck Group (1 mentions) Glutathione gsh reduced free acid, by Merck Group (1 mentions) Nr4a1 antibody, by Novus Biologicals (1 mentions) P fak, by Cell Signaling Technology (1 mentions) Panc 1, by American Type Culture Collection (1 mentions) Mia paca 2, by American Type Culture Collection (1 mentions) Survivin antibody, by Cell Signaling Technology (1 mentions) Rpmi 1640 medium, by Merck Group (1 mentions) Sw480, by American Type Culture Collection (1 mentions)

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Anti-SP1 antibody (12 citations)

6 protocols using sp1 antibody

1

ChIP-seq protocol for SP1 transcription factor

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B cells were isolated by negative selection using the human B cell isolation kit II (Miltenyi). Cells were fixed with 4% paraformaldehyde for 10 minutes at 37°C. The cell pellet was frozen at −80°C until sample preparation. Magnetic beads were blocked (PBS/0.5% BSA) and incubated with either 10 μg SP1 antibody (07-645, Millipore) or control IgG overnight at 4°C. Nuclei were isolated the following day and sheared using a truChIP Chromatin Shearing Reagent Kit (Covaris), an AFA microTUBE (Covaris) and a M220 focused ultrasonicator (Covaris). Conditions for sonication: 20% duty cycle, 30 min. Sonicated samples were immunoprecipitated with magnetically labeled SP1 antibody or control IgG overnight at 4°C. The next day samples were reverse cross-linked. The Qiagen QIAquick PCR purification kit was used to purify the DNA.
Dam E.M., Maier A.C., Hocking A.M., Carlin J., Ng B, & Buckner J.H. (2018). Increased Binding of Specificity Protein 1 to the IL21R Promoter in B Cells Results in Enhanced B Cell Responses in Rheumatoid Arthritis. Frontiers in Immunology, 9, 1978.
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2

ChIP Assay of Sp1, Sp3, and RNA Pol II Binding

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Chromatin immunoprecipitation (ChIP) assay was performed using Pierce Agarose ChIP Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s protocol. HCT116 and HepG2 cells were cultured with or without TSA (1 μM) for 24 hours. The DNA-protein complexes were immunoprecipitated with 8 μg of Sp1 antibody (Millipore, USA), 8 μg of Sp3 antibody (Millipore, USA) and 8 μg of RNA polymerase II (RNA pol II) antibody (Thermo Fisher Scientific, USA). DNA recovered from samples was PCR amplified using ek1-154-5’ and ek1-3’ primers followed by agarose gel electrophoresis. The intensities of the bands of interest were quantitated with Image J 1.42 program [23 (link)]. All the PCR products were gel-purified and sequenced to confirm that the PCR products were ek1 promoter.
Kuan C.S., See Too W.C, & Few L.L. (2016). Sp1 and Sp3 Are the Transcription Activators of Human ek1 Promoter in TSA-Treated Human Colon Carcinoma Cells. PLoS ONE, 11(1), e0147886.
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3

STAT-binding Validation via qPCR

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To validate STAT-binding to promoter regions, Taqman custom assays were designed (see Supplementary Table 2 for oligonucleotide primer sequences) and qPCR performed using a QuantStudio 12K Flex Real-Time PCR System. For analysis of SP1, chromatin immunoprecipitation was conducted as previously described using 5ug of SP1 antibody (#17-601, Millipore) or isotype specific IgG control. Analysis by qPCR used oligonucleotide primer sequences to the promoter regions of Irf1, Socs3, Stat3, Cd274, Il4ra, Junb and Socs1 (Supplementary Table 2). Specific enrichment was normalised by subtracting the IgG control values from those derived for the input and antibody specific immunoprecipitation samples. Value were expressed as 2^ΔCT.
Twohig J.P., Figueras A.C., Andrews R., Wiede F., Cossins B.C., Soria A.D., Lewis M.J., Townsend M.J., Millrine D., Li J., Hill D.G., Fernandez J.U., Liu X., Szomolay B., Pepper C.J., Taylor P.R., Pitzalis C., Tiganis T., Williams N.M., Jones G.W, & Jones S.A. (2019). Activation of naïve CD4+ T cells re-tunes STAT1 signaling to deliver unique cytokine responses in memory CD4+ T cells. Nature immunology, 20(4), 458-470.
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4

Molecular Mechanisms of Pancreatic and Colon Cancer

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Pancreatic cancer (Panc1 and MiaPaCa2) and colon cancer (RKO, SW480) cell lines were purchased from American Type Culture Collection (Manassas, VA) and were authenticated on April 29, 2016 by Biosynthesis (Lewisville, TX). L3.6pL cells were kindly provided by Dr. I.J. Fidler (University of Texas MD Anderson Cancer Center, Houston, TX). Cells were maintained 37°C in the presence of 5% CO2 in Dulbecco’s Modified Eagle’s Medium/Ham’s F-12 medium with 10% fetal bovine serum with antibiotic. NR4A1 antibody was purchased from Novus Biologicals (Littleton, CO). Sp1 antibody and glutathione (GSH) reduced free acid were purchased from Millipore (Temecula, CA); β1-integrin, p300, Sp3 and Sp4 antibodies were purchased from Santa Cruz Biotech (Santa Cruz, CA). β-Actin antibody, Dulbecco’s Modified Eagle’s Medium, and 36% formaldehyde were purchased from Sigma-Aldrich (St. Louis, MO). Hematoxylin was purchased from Vector Laboratories (Burlingame, CA). FAK, p-FAK, α5-integrin antibodies and leptomycin were purchased from Cell Signaling Technologies (Manassas, VA).
Hedrick E., Lee S.O, & Safe S. (2017). The Nuclear Orphan Receptor NR4A1 Regulates β1-Integrin Expression in Pancreatic and Colon Cancer Cells and Can Be Targeted by NR4A1 Antagonists. Molecular carcinogenesis, 56(9), 2066-2075.
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5

STAT-binding Validation via qPCR

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To validate STAT-binding to promoter regions, Taqman custom assays were designed (see Supplementary Table 2 for oligonucleotide primer sequences) and qPCR performed using a QuantStudio 12K Flex Real-Time PCR System. For analysis of SP1, chromatin immunoprecipitation was conducted as previously described using 5ug of SP1 antibody (#17-601, Millipore) or isotype specific IgG control. Analysis by qPCR used oligonucleotide primer sequences to the promoter regions of Irf1, Socs3, Stat3, Cd274, Il4ra, Junb and Socs1 (Supplementary Table 2). Specific enrichment was normalised by subtracting the IgG control values from those derived for the input and antibody specific immunoprecipitation samples. Value were expressed as 2^ΔCT.
Twohig J.P., Figueras A.C., Andrews R., Wiede F., Cossins B.C., Soria A.D., Lewis M.J., Townsend M.J., Millrine D., Li J., Hill D.G., Fernandez J.U., Liu X., Szomolay B., Pepper C.J., Taylor P.R., Pitzalis C., Tiganis T., Williams N.M., Jones G.W, & Jones S.A. (2019). Activation of naïve CD4+ T cells re-tunes STAT1 signaling to deliver unique cytokine responses in memory CD4+ T cells. Nature immunology, 20(4), 458-470.
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6

Profiling Cancer Cell Lines

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Breast (SKBR3, MDA-MB-231), kidney (786-O), colorectal cancer (SW480), lung (A549), and pancreatic (Panc1, L3.6pL, MiaPaCa2) cancer cell lines were purchased from American Type Culture Collection (Manassas, VA). Cells were maintained 37°C in the presence of 5% CO2 in Dulbecco's Modified Eagle's Medium/Ham's F-12 medium with 10% fetal bovine serum with antibiotic or RPMI-1640 Medium with 10% fetal bovine serum and antibiotic. b-Actin antibody, Dulbecco's Modified Eagle's Medium, and RPMI-1640 Medium, and 36% formaldehyde were purchased from Sigma-Aldrich (St. Louis, MO). Hematoxylin was purchased from Vector Laboratories (Burlingame, CA). Sp1 antibody from Millipore (Temecula, CA); Sp3, Sp4, EGFR, bcl2 antibodies from Santa Cruz Biotech (Santa Cruz, CA); survivin antibody from Cell Signaling Technologies (Danvers, MA); VEGF antibody from GeneTex (Irvine, CA). Apoptotic, Necrotic, and Healthy Cells Quantification Kit was purchased from Biotium (Hayward, CA). Cells were visualized as described previously [21 (link)].
Hedrick E., Cheng Y., Jin U.H., Kim K, & Safe S. (2016). Specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 are non-oncogene addiction genes in cancer cells. Oncotarget, 7(16), 22245-22256.
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