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Mutp53 binds VDR and augments the transcriptional response of classical VDR target genes to vitamin D3 A. SKBR3 and MCF7 cells were either grown in charcoal-stripped serum supplemented medium (-D3) or treated with 1α25(OH) 2 . B. H1299 cells were transfected with a VDR expression plasmid together with plasmids expressing either full length H175Rp53, ΔN-H175Rp53 (aa 97-393), ΔC-H175Rp53 (aa 1-292) or the <t>DNA</t> binding domain of H175Rp53 (DBD; aa 97-292). Total protein input levels are shown in the left panel. Cells were treated with 1α25(OH) 2 D3 for 3h and cell extracts were subjected to immunoprecipitation with either anti VDR C-20 (Santa Cruz) or PAb419 as a non-specific control (ns-Ab). Immunoprecipitated proteins were analyzed as in (A). C. SKBR3 and SW480 cells were transfected with siRNA oligonucleotides specific for p53 (p53i) or LacZ (LacZi) as negative control. After 24h, 1α25(OH) 2 D3 (D3, 100nM) was added for 24h where indicated. Cells were harvested, RNA was extracted and <t>qRT-PCR</t> was performed with primers specific for IGFBP3 , CYP24A1 or GAPDH . Relative levels of IGFBP3 and CYP24A1 mRNA were normalized to GAPDH . D. SKBR3 cells were transfected with either p53 siRNA (sip53) or control siRNA (si control) and treated with 1α25(OH) 2 D3 (D3;100nM, 2hours) or left untreated. Cells were subjected to chromatin immunoprecipitation with antibodies directed against either p53, VDR, p300 or non-specific antibody (ns). The precipitated DNA was subjected to Real-time qPCR amplification using sets of primers specific to a region of the CYP24A promoter spanning the VDRE. Results are presented as fold over non-specific antibody. E. MDA-MB-231 cells grown in medium containing charcoal-stripped serum, or treated with 1α25(OH)2D3 (gray bars, D3;100nM, 2 hours) or left untreated (white bars), and then subjected to chromatin immunoprecipitation with antibodies directed against p53. The immnoprecipitated chromatin was eluted and then a second IP was performed with antibodies against VDR, p300 or non specific antibody (ns Ab) as a negative control. Real-time qPCR was performed as in (D). F. RNA was prepared from the experiment shown in panel C and subjected to qRT-PCR analysis with primers specific for CYP27B . G. Immunoprecipitated DNA from experiment shown in (D) was subjected to Real-time PCR amplification with primers specific to a region of the CYP27B promoter spanning the nVDRE. Results are presented as fold over non-specific antibody. All scale bars represent +/-SD.
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1) Product Images from "Modulation of the Vitamin D3 response by cancer-associated mutant p53"

Article Title: Modulation of the Vitamin D3 response by cancer-associated mutant p53

Journal: Cancer cell

doi: 10.1016/j.ccr.2009.11.025

Mutp53 binds VDR and augments the transcriptional response of classical VDR target genes to vitamin D3 A. SKBR3 and MCF7 cells were either grown in charcoal-stripped serum supplemented medium (-D3) or treated with 1α25(OH) 2 . B. H1299 cells were transfected with a VDR expression plasmid together with plasmids expressing either full length H175Rp53, ΔN-H175Rp53 (aa 97-393), ΔC-H175Rp53 (aa 1-292) or the DNA binding domain of H175Rp53 (DBD; aa 97-292). Total protein input levels are shown in the left panel. Cells were treated with 1α25(OH) 2 D3 for 3h and cell extracts were subjected to immunoprecipitation with either anti VDR C-20 (Santa Cruz) or PAb419 as a non-specific control (ns-Ab). Immunoprecipitated proteins were analyzed as in (A). C. SKBR3 and SW480 cells were transfected with siRNA oligonucleotides specific for p53 (p53i) or LacZ (LacZi) as negative control. After 24h, 1α25(OH) 2 D3 (D3, 100nM) was added for 24h where indicated. Cells were harvested, RNA was extracted and qRT-PCR was performed with primers specific for IGFBP3 , CYP24A1 or GAPDH . Relative levels of IGFBP3 and CYP24A1 mRNA were normalized to GAPDH . D. SKBR3 cells were transfected with either p53 siRNA (sip53) or control siRNA (si control) and treated with 1α25(OH) 2 D3 (D3;100nM, 2hours) or left untreated. Cells were subjected to chromatin immunoprecipitation with antibodies directed against either p53, VDR, p300 or non-specific antibody (ns). The precipitated DNA was subjected to Real-time qPCR amplification using sets of primers specific to a region of the CYP24A promoter spanning the VDRE. Results are presented as fold over non-specific antibody. E. MDA-MB-231 cells grown in medium containing charcoal-stripped serum, or treated with 1α25(OH)2D3 (gray bars, D3;100nM, 2 hours) or left untreated (white bars), and then subjected to chromatin immunoprecipitation with antibodies directed against p53. The immnoprecipitated chromatin was eluted and then a second IP was performed with antibodies against VDR, p300 or non specific antibody (ns Ab) as a negative control. Real-time qPCR was performed as in (D). F. RNA was prepared from the experiment shown in panel C and subjected to qRT-PCR analysis with primers specific for CYP27B . G. Immunoprecipitated DNA from experiment shown in (D) was subjected to Real-time PCR amplification with primers specific to a region of the CYP27B promoter spanning the nVDRE. Results are presented as fold over non-specific antibody. All scale bars represent +/-SD.
Figure Legend Snippet: Mutp53 binds VDR and augments the transcriptional response of classical VDR target genes to vitamin D3 A. SKBR3 and MCF7 cells were either grown in charcoal-stripped serum supplemented medium (-D3) or treated with 1α25(OH) 2 . B. H1299 cells were transfected with a VDR expression plasmid together with plasmids expressing either full length H175Rp53, ΔN-H175Rp53 (aa 97-393), ΔC-H175Rp53 (aa 1-292) or the DNA binding domain of H175Rp53 (DBD; aa 97-292). Total protein input levels are shown in the left panel. Cells were treated with 1α25(OH) 2 D3 for 3h and cell extracts were subjected to immunoprecipitation with either anti VDR C-20 (Santa Cruz) or PAb419 as a non-specific control (ns-Ab). Immunoprecipitated proteins were analyzed as in (A). C. SKBR3 and SW480 cells were transfected with siRNA oligonucleotides specific for p53 (p53i) or LacZ (LacZi) as negative control. After 24h, 1α25(OH) 2 D3 (D3, 100nM) was added for 24h where indicated. Cells were harvested, RNA was extracted and qRT-PCR was performed with primers specific for IGFBP3 , CYP24A1 or GAPDH . Relative levels of IGFBP3 and CYP24A1 mRNA were normalized to GAPDH . D. SKBR3 cells were transfected with either p53 siRNA (sip53) or control siRNA (si control) and treated with 1α25(OH) 2 D3 (D3;100nM, 2hours) or left untreated. Cells were subjected to chromatin immunoprecipitation with antibodies directed against either p53, VDR, p300 or non-specific antibody (ns). The precipitated DNA was subjected to Real-time qPCR amplification using sets of primers specific to a region of the CYP24A promoter spanning the VDRE. Results are presented as fold over non-specific antibody. E. MDA-MB-231 cells grown in medium containing charcoal-stripped serum, or treated with 1α25(OH)2D3 (gray bars, D3;100nM, 2 hours) or left untreated (white bars), and then subjected to chromatin immunoprecipitation with antibodies directed against p53. The immnoprecipitated chromatin was eluted and then a second IP was performed with antibodies against VDR, p300 or non specific antibody (ns Ab) as a negative control. Real-time qPCR was performed as in (D). F. RNA was prepared from the experiment shown in panel C and subjected to qRT-PCR analysis with primers specific for CYP27B . G. Immunoprecipitated DNA from experiment shown in (D) was subjected to Real-time PCR amplification with primers specific to a region of the CYP27B promoter spanning the nVDRE. Results are presented as fold over non-specific antibody. All scale bars represent +/-SD.

Techniques Used: Transfection, Expressing, Plasmid Preparation, Binding Assay, Immunoprecipitation, Negative Control, Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Amplification, Multiple Displacement Amplification

Mutp53 associates with VDR/RXR response elements and augments transcription from such elements A. SKBR3 cells expressing p53R175H, transfected with either siRNA for p53 (sip53) or control siRNA (sicontrol) and treated with 1α25(OH) 2 D3 (D3;100nM), were subjected to chromatin immunoprecipitation with an antibodies against p53 or VDR or with a non specific antibody as a negative control. The precipitated DNA was subjected to Real-time PCR amplification using sets of primers specific to either ChIP-control (negative control), or regions of the HIRA and TGFβ1 gene promoters spanning putative VDR/RXR response elements (VDRE). Results are presented relative to the values obtained with an aliquot of the input chromatin corresponding to 1% of the total material. The results of HIRA and TGFβ1 gene promoters are presented as enrichment fold over non specific antibody and are also normalized to values obtained for the ChIP-control results. B. Reporter plasmids were either cotransfected together with mutp53 expression plasmids (R175H or R273H) into p53-null H1299 cells (upper panel) or cotransfected with synthetic siRNA specific for p53 (p53i) or LacZ (LacZi) into SKBR3 cells (bottom panel). Cells were either treated with 1α25(OH) 2 . All scale bars represent +/-SD.
Figure Legend Snippet: Mutp53 associates with VDR/RXR response elements and augments transcription from such elements A. SKBR3 cells expressing p53R175H, transfected with either siRNA for p53 (sip53) or control siRNA (sicontrol) and treated with 1α25(OH) 2 D3 (D3;100nM), were subjected to chromatin immunoprecipitation with an antibodies against p53 or VDR or with a non specific antibody as a negative control. The precipitated DNA was subjected to Real-time PCR amplification using sets of primers specific to either ChIP-control (negative control), or regions of the HIRA and TGFβ1 gene promoters spanning putative VDR/RXR response elements (VDRE). Results are presented relative to the values obtained with an aliquot of the input chromatin corresponding to 1% of the total material. The results of HIRA and TGFβ1 gene promoters are presented as enrichment fold over non specific antibody and are also normalized to values obtained for the ChIP-control results. B. Reporter plasmids were either cotransfected together with mutp53 expression plasmids (R175H or R273H) into p53-null H1299 cells (upper panel) or cotransfected with synthetic siRNA specific for p53 (p53i) or LacZ (LacZi) into SKBR3 cells (bottom panel). Cells were either treated with 1α25(OH) 2 . All scale bars represent +/-SD.

Techniques Used: Expressing, Transfection, Chromatin Immunoprecipitation, Negative Control, Real-time Polymerase Chain Reaction, Amplification

Related Articles

Polymerase Chain Reaction:

Article Title: Modulation of the Vitamin D3 response by cancer-associated mutant p53
Article Snippet: .. DNA samples were extracted using PCR clean-up mini-columns (Promega). ..

Chromatin Immunoprecipitation:

Article Title: Modulation of the Vitamin D3 response by cancer-associated mutant p53
Article Snippet: Paragraph title: Chromatin immunoprecipitation ... DNA samples were extracted using PCR clean-up mini-columns (Promega).

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    Promega pcr clean up mini columns
    Mutp53 binds VDR and augments the transcriptional response of classical VDR target genes to vitamin D3 A. SKBR3 and MCF7 cells were either grown in charcoal-stripped serum supplemented medium (-D3) or treated with 1α25(OH) 2 . B. H1299 cells were transfected with a VDR expression plasmid together with plasmids expressing either full length H175Rp53, ΔN-H175Rp53 (aa 97-393), ΔC-H175Rp53 (aa 1-292) or the <t>DNA</t> binding domain of H175Rp53 (DBD; aa 97-292). Total protein input levels are shown in the left panel. Cells were treated with 1α25(OH) 2 D3 for 3h and cell extracts were subjected to immunoprecipitation with either anti VDR C-20 (Santa Cruz) or PAb419 as a non-specific control (ns-Ab). Immunoprecipitated proteins were analyzed as in (A). C. SKBR3 and SW480 cells were transfected with siRNA oligonucleotides specific for p53 (p53i) or LacZ (LacZi) as negative control. After 24h, 1α25(OH) 2 D3 (D3, 100nM) was added for 24h where indicated. Cells were harvested, RNA was extracted and <t>qRT-PCR</t> was performed with primers specific for IGFBP3 , CYP24A1 or GAPDH . Relative levels of IGFBP3 and CYP24A1 mRNA were normalized to GAPDH . D. SKBR3 cells were transfected with either p53 siRNA (sip53) or control siRNA (si control) and treated with 1α25(OH) 2 D3 (D3;100nM, 2hours) or left untreated. Cells were subjected to chromatin immunoprecipitation with antibodies directed against either p53, VDR, p300 or non-specific antibody (ns). The precipitated DNA was subjected to Real-time qPCR amplification using sets of primers specific to a region of the CYP24A promoter spanning the VDRE. Results are presented as fold over non-specific antibody. E. MDA-MB-231 cells grown in medium containing charcoal-stripped serum, or treated with 1α25(OH)2D3 (gray bars, D3;100nM, 2 hours) or left untreated (white bars), and then subjected to chromatin immunoprecipitation with antibodies directed against p53. The immnoprecipitated chromatin was eluted and then a second IP was performed with antibodies against VDR, p300 or non specific antibody (ns Ab) as a negative control. Real-time qPCR was performed as in (D). F. RNA was prepared from the experiment shown in panel C and subjected to qRT-PCR analysis with primers specific for CYP27B . G. Immunoprecipitated DNA from experiment shown in (D) was subjected to Real-time PCR amplification with primers specific to a region of the CYP27B promoter spanning the nVDRE. Results are presented as fold over non-specific antibody. All scale bars represent +/-SD.
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    Mutp53 binds VDR and augments the transcriptional response of classical VDR target genes to vitamin D3 A. SKBR3 and MCF7 cells were either grown in charcoal-stripped serum supplemented medium (-D3) or treated with 1α25(OH) 2 . B. H1299 cells were transfected with a VDR expression plasmid together with plasmids expressing either full length H175Rp53, ΔN-H175Rp53 (aa 97-393), ΔC-H175Rp53 (aa 1-292) or the <t>DNA</t> binding domain of H175Rp53 (DBD; aa 97-292). Total protein input levels are shown in the left panel. Cells were treated with 1α25(OH) 2 D3 for 3h and cell extracts were subjected to immunoprecipitation with either anti VDR C-20 (Santa Cruz) or PAb419 as a non-specific control (ns-Ab). Immunoprecipitated proteins were analyzed as in (A). C. SKBR3 and SW480 cells were transfected with siRNA oligonucleotides specific for p53 (p53i) or LacZ (LacZi) as negative control. After 24h, 1α25(OH) 2 D3 (D3, 100nM) was added for 24h where indicated. Cells were harvested, RNA was extracted and <t>qRT-PCR</t> was performed with primers specific for IGFBP3 , CYP24A1 or GAPDH . Relative levels of IGFBP3 and CYP24A1 mRNA were normalized to GAPDH . D. SKBR3 cells were transfected with either p53 siRNA (sip53) or control siRNA (si control) and treated with 1α25(OH) 2 D3 (D3;100nM, 2hours) or left untreated. Cells were subjected to chromatin immunoprecipitation with antibodies directed against either p53, VDR, p300 or non-specific antibody (ns). The precipitated DNA was subjected to Real-time qPCR amplification using sets of primers specific to a region of the CYP24A promoter spanning the VDRE. Results are presented as fold over non-specific antibody. E. MDA-MB-231 cells grown in medium containing charcoal-stripped serum, or treated with 1α25(OH)2D3 (gray bars, D3;100nM, 2 hours) or left untreated (white bars), and then subjected to chromatin immunoprecipitation with antibodies directed against p53. The immnoprecipitated chromatin was eluted and then a second IP was performed with antibodies against VDR, p300 or non specific antibody (ns Ab) as a negative control. Real-time qPCR was performed as in (D). F. RNA was prepared from the experiment shown in panel C and subjected to qRT-PCR analysis with primers specific for CYP27B . G. Immunoprecipitated DNA from experiment shown in (D) was subjected to Real-time PCR amplification with primers specific to a region of the CYP27B promoter spanning the nVDRE. Results are presented as fold over non-specific antibody. All scale bars represent +/-SD.
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    Mutp53 binds VDR and augments the transcriptional response of classical VDR target genes to vitamin D3 A. SKBR3 and MCF7 cells were either grown in charcoal-stripped serum supplemented medium (-D3) or treated with 1α25(OH) 2 . B. H1299 cells were transfected with a VDR expression plasmid together with plasmids expressing either full length H175Rp53, ΔN-H175Rp53 (aa 97-393), ΔC-H175Rp53 (aa 1-292) or the DNA binding domain of H175Rp53 (DBD; aa 97-292). Total protein input levels are shown in the left panel. Cells were treated with 1α25(OH) 2 D3 for 3h and cell extracts were subjected to immunoprecipitation with either anti VDR C-20 (Santa Cruz) or PAb419 as a non-specific control (ns-Ab). Immunoprecipitated proteins were analyzed as in (A). C. SKBR3 and SW480 cells were transfected with siRNA oligonucleotides specific for p53 (p53i) or LacZ (LacZi) as negative control. After 24h, 1α25(OH) 2 D3 (D3, 100nM) was added for 24h where indicated. Cells were harvested, RNA was extracted and qRT-PCR was performed with primers specific for IGFBP3 , CYP24A1 or GAPDH . Relative levels of IGFBP3 and CYP24A1 mRNA were normalized to GAPDH . D. SKBR3 cells were transfected with either p53 siRNA (sip53) or control siRNA (si control) and treated with 1α25(OH) 2 D3 (D3;100nM, 2hours) or left untreated. Cells were subjected to chromatin immunoprecipitation with antibodies directed against either p53, VDR, p300 or non-specific antibody (ns). The precipitated DNA was subjected to Real-time qPCR amplification using sets of primers specific to a region of the CYP24A promoter spanning the VDRE. Results are presented as fold over non-specific antibody. E. MDA-MB-231 cells grown in medium containing charcoal-stripped serum, or treated with 1α25(OH)2D3 (gray bars, D3;100nM, 2 hours) or left untreated (white bars), and then subjected to chromatin immunoprecipitation with antibodies directed against p53. The immnoprecipitated chromatin was eluted and then a second IP was performed with antibodies against VDR, p300 or non specific antibody (ns Ab) as a negative control. Real-time qPCR was performed as in (D). F. RNA was prepared from the experiment shown in panel C and subjected to qRT-PCR analysis with primers specific for CYP27B . G. Immunoprecipitated DNA from experiment shown in (D) was subjected to Real-time PCR amplification with primers specific to a region of the CYP27B promoter spanning the nVDRE. Results are presented as fold over non-specific antibody. All scale bars represent +/-SD.

    Journal: Cancer cell

    Article Title: Modulation of the Vitamin D3 response by cancer-associated mutant p53

    doi: 10.1016/j.ccr.2009.11.025

    Figure Lengend Snippet: Mutp53 binds VDR and augments the transcriptional response of classical VDR target genes to vitamin D3 A. SKBR3 and MCF7 cells were either grown in charcoal-stripped serum supplemented medium (-D3) or treated with 1α25(OH) 2 . B. H1299 cells were transfected with a VDR expression plasmid together with plasmids expressing either full length H175Rp53, ΔN-H175Rp53 (aa 97-393), ΔC-H175Rp53 (aa 1-292) or the DNA binding domain of H175Rp53 (DBD; aa 97-292). Total protein input levels are shown in the left panel. Cells were treated with 1α25(OH) 2 D3 for 3h and cell extracts were subjected to immunoprecipitation with either anti VDR C-20 (Santa Cruz) or PAb419 as a non-specific control (ns-Ab). Immunoprecipitated proteins were analyzed as in (A). C. SKBR3 and SW480 cells were transfected with siRNA oligonucleotides specific for p53 (p53i) or LacZ (LacZi) as negative control. After 24h, 1α25(OH) 2 D3 (D3, 100nM) was added for 24h where indicated. Cells were harvested, RNA was extracted and qRT-PCR was performed with primers specific for IGFBP3 , CYP24A1 or GAPDH . Relative levels of IGFBP3 and CYP24A1 mRNA were normalized to GAPDH . D. SKBR3 cells were transfected with either p53 siRNA (sip53) or control siRNA (si control) and treated with 1α25(OH) 2 D3 (D3;100nM, 2hours) or left untreated. Cells were subjected to chromatin immunoprecipitation with antibodies directed against either p53, VDR, p300 or non-specific antibody (ns). The precipitated DNA was subjected to Real-time qPCR amplification using sets of primers specific to a region of the CYP24A promoter spanning the VDRE. Results are presented as fold over non-specific antibody. E. MDA-MB-231 cells grown in medium containing charcoal-stripped serum, or treated with 1α25(OH)2D3 (gray bars, D3;100nM, 2 hours) or left untreated (white bars), and then subjected to chromatin immunoprecipitation with antibodies directed against p53. The immnoprecipitated chromatin was eluted and then a second IP was performed with antibodies against VDR, p300 or non specific antibody (ns Ab) as a negative control. Real-time qPCR was performed as in (D). F. RNA was prepared from the experiment shown in panel C and subjected to qRT-PCR analysis with primers specific for CYP27B . G. Immunoprecipitated DNA from experiment shown in (D) was subjected to Real-time PCR amplification with primers specific to a region of the CYP27B promoter spanning the nVDRE. Results are presented as fold over non-specific antibody. All scale bars represent +/-SD.

    Article Snippet: DNA samples were extracted using PCR clean-up mini-columns (Promega).

    Techniques: Transfection, Expressing, Plasmid Preparation, Binding Assay, Immunoprecipitation, Negative Control, Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Amplification, Multiple Displacement Amplification

    Mutp53 associates with VDR/RXR response elements and augments transcription from such elements A. SKBR3 cells expressing p53R175H, transfected with either siRNA for p53 (sip53) or control siRNA (sicontrol) and treated with 1α25(OH) 2 D3 (D3;100nM), were subjected to chromatin immunoprecipitation with an antibodies against p53 or VDR or with a non specific antibody as a negative control. The precipitated DNA was subjected to Real-time PCR amplification using sets of primers specific to either ChIP-control (negative control), or regions of the HIRA and TGFβ1 gene promoters spanning putative VDR/RXR response elements (VDRE). Results are presented relative to the values obtained with an aliquot of the input chromatin corresponding to 1% of the total material. The results of HIRA and TGFβ1 gene promoters are presented as enrichment fold over non specific antibody and are also normalized to values obtained for the ChIP-control results. B. Reporter plasmids were either cotransfected together with mutp53 expression plasmids (R175H or R273H) into p53-null H1299 cells (upper panel) or cotransfected with synthetic siRNA specific for p53 (p53i) or LacZ (LacZi) into SKBR3 cells (bottom panel). Cells were either treated with 1α25(OH) 2 . All scale bars represent +/-SD.

    Journal: Cancer cell

    Article Title: Modulation of the Vitamin D3 response by cancer-associated mutant p53

    doi: 10.1016/j.ccr.2009.11.025

    Figure Lengend Snippet: Mutp53 associates with VDR/RXR response elements and augments transcription from such elements A. SKBR3 cells expressing p53R175H, transfected with either siRNA for p53 (sip53) or control siRNA (sicontrol) and treated with 1α25(OH) 2 D3 (D3;100nM), were subjected to chromatin immunoprecipitation with an antibodies against p53 or VDR or with a non specific antibody as a negative control. The precipitated DNA was subjected to Real-time PCR amplification using sets of primers specific to either ChIP-control (negative control), or regions of the HIRA and TGFβ1 gene promoters spanning putative VDR/RXR response elements (VDRE). Results are presented relative to the values obtained with an aliquot of the input chromatin corresponding to 1% of the total material. The results of HIRA and TGFβ1 gene promoters are presented as enrichment fold over non specific antibody and are also normalized to values obtained for the ChIP-control results. B. Reporter plasmids were either cotransfected together with mutp53 expression plasmids (R175H or R273H) into p53-null H1299 cells (upper panel) or cotransfected with synthetic siRNA specific for p53 (p53i) or LacZ (LacZi) into SKBR3 cells (bottom panel). Cells were either treated with 1α25(OH) 2 . All scale bars represent +/-SD.

    Article Snippet: DNA samples were extracted using PCR clean-up mini-columns (Promega).

    Techniques: Expressing, Transfection, Chromatin Immunoprecipitation, Negative Control, Real-time Polymerase Chain Reaction, Amplification