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flag tagged human klf4 expression vectors  (OriGene)


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    OriGene flag tagged human klf4 expression vectors
    A ) PU/ER(T) cells were grown in presence of 95% Ethanol or 100 nM Tamoxifen for time periods as indicated. Cytosolic and nuclear protein fractions were prepared and translocation of PU.1 from cytosol to nucleus was detected by immunoblotting with anti PU.1 rabbit polyclonal antibodies. Purity of cytoplasmic and nuclear fractions was analyzed by immunoblotting with α-Tubulin and HDAC2 proteins. B ) Nuclear extracts were prepared from differentiating bone marrow derived macrophages on day 1 and 7 and in vitro binding to biotin labeled 76 bp oligonucleotide probe representing −118/−113 PU.1 binding element in the <t>KLF4</t> promoter was determined by EMSA as described in . C ) PU/ER(T) cells were treated with Ethanol or 100 nM Tamoxifen for 1, 4 or 24 h and nuclear extracts were prepared. An equal amount of nuclear protein extract from Ethanol and Tamoxifen treatment was analyzed for DNA binding activity using biotin labeled 76 bp oligonucleotide probe representing −118/−113 PU.1 binding element in the KLF4 promoter. Specificity of PU.1-KLF4 DNA complex in experiment B & C was determined by the ability of the PU.1-KLF4 DNA complex to form supershift with anti PU.1 antibody. D ) Specificity of PU.1-KLF4 DNA complex was determined by including 5, 15, 25 and 100 picomole of excess unlabelled KLF4 promoter oligo in the DNA binding reaction. E ) In vivo binding of PU.1 to KLF4 promoter was determined by Chromatin-immuno precipitation in differentiating BMDM on day 1, 2 and 7 and in F ) PU/ER(T) cells treated with Tamoxifen for 1 and 24 h. Error bars represent standard deviation and * is used where ever p value is ≤0.05 and ** is used when p≤0.005.
    Flag Tagged Human Klf4 Expression Vectors, supplied by OriGene, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/flag+tagged+human+klf4+expression+vectors/pmc03973678-46-4-13?v=OriGene
    Average 90 stars, based on 1 article reviews
    flag tagged human klf4 expression vectors - by Bioz Stars, 2026-08
    90/100 stars

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    1) Product Images from "Krüppel Like Factor 4 Promoter Undergoes Active Demethylation during Monocyte/Macrophage Differentiation"

    Article Title: Krüppel Like Factor 4 Promoter Undergoes Active Demethylation during Monocyte/Macrophage Differentiation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0093362

    A ) PU/ER(T) cells were grown in presence of 95% Ethanol or 100 nM Tamoxifen for time periods as indicated. Cytosolic and nuclear protein fractions were prepared and translocation of PU.1 from cytosol to nucleus was detected by immunoblotting with anti PU.1 rabbit polyclonal antibodies. Purity of cytoplasmic and nuclear fractions was analyzed by immunoblotting with α-Tubulin and HDAC2 proteins. B ) Nuclear extracts were prepared from differentiating bone marrow derived macrophages on day 1 and 7 and in vitro binding to biotin labeled 76 bp oligonucleotide probe representing −118/−113 PU.1 binding element in the KLF4 promoter was determined by EMSA as described in . C ) PU/ER(T) cells were treated with Ethanol or 100 nM Tamoxifen for 1, 4 or 24 h and nuclear extracts were prepared. An equal amount of nuclear protein extract from Ethanol and Tamoxifen treatment was analyzed for DNA binding activity using biotin labeled 76 bp oligonucleotide probe representing −118/−113 PU.1 binding element in the KLF4 promoter. Specificity of PU.1-KLF4 DNA complex in experiment B & C was determined by the ability of the PU.1-KLF4 DNA complex to form supershift with anti PU.1 antibody. D ) Specificity of PU.1-KLF4 DNA complex was determined by including 5, 15, 25 and 100 picomole of excess unlabelled KLF4 promoter oligo in the DNA binding reaction. E ) In vivo binding of PU.1 to KLF4 promoter was determined by Chromatin-immuno precipitation in differentiating BMDM on day 1, 2 and 7 and in F ) PU/ER(T) cells treated with Tamoxifen for 1 and 24 h. Error bars represent standard deviation and * is used where ever p value is ≤0.05 and ** is used when p≤0.005.
    Figure Legend Snippet: A ) PU/ER(T) cells were grown in presence of 95% Ethanol or 100 nM Tamoxifen for time periods as indicated. Cytosolic and nuclear protein fractions were prepared and translocation of PU.1 from cytosol to nucleus was detected by immunoblotting with anti PU.1 rabbit polyclonal antibodies. Purity of cytoplasmic and nuclear fractions was analyzed by immunoblotting with α-Tubulin and HDAC2 proteins. B ) Nuclear extracts were prepared from differentiating bone marrow derived macrophages on day 1 and 7 and in vitro binding to biotin labeled 76 bp oligonucleotide probe representing −118/−113 PU.1 binding element in the KLF4 promoter was determined by EMSA as described in . C ) PU/ER(T) cells were treated with Ethanol or 100 nM Tamoxifen for 1, 4 or 24 h and nuclear extracts were prepared. An equal amount of nuclear protein extract from Ethanol and Tamoxifen treatment was analyzed for DNA binding activity using biotin labeled 76 bp oligonucleotide probe representing −118/−113 PU.1 binding element in the KLF4 promoter. Specificity of PU.1-KLF4 DNA complex in experiment B & C was determined by the ability of the PU.1-KLF4 DNA complex to form supershift with anti PU.1 antibody. D ) Specificity of PU.1-KLF4 DNA complex was determined by including 5, 15, 25 and 100 picomole of excess unlabelled KLF4 promoter oligo in the DNA binding reaction. E ) In vivo binding of PU.1 to KLF4 promoter was determined by Chromatin-immuno precipitation in differentiating BMDM on day 1, 2 and 7 and in F ) PU/ER(T) cells treated with Tamoxifen for 1 and 24 h. Error bars represent standard deviation and * is used where ever p value is ≤0.05 and ** is used when p≤0.005.

    Techniques Used: Translocation Assay, Western Blot, Derivative Assay, In Vitro, Binding Assay, Labeling, Activity Assay, In Vivo, Chromatin Immunoprecipitation, Standard Deviation

    A ) Methprimer analysis of KLF4 promoter indicating dense CpG methylation distributed as three distinct regions, M1CpG proximal to the transcription start site and two distal M2 and M3CpG regions away from the transcription start site. B ) TF search analysis of 1.6 kb KLF4 promoter indicating putative PU.1 binding elements at −113 and −35 bp region from the transcription start site. C ) Total genomic DNA was isolated, Bisulfite converted and used as template for qPCR using methylation specific primers. The relative methylation index of M1, M2 and M3 CpG regions calculated as described in was compared in PUER(T) cells after treatment with Ethanol or Tamoxifen for 72 h and in D ) differentiating BMDM on 1 st and 7 th days E ) Methylation of M1CpG region was compared by bisulfite sequencing in control and 72 h 4-OHT treated PU/ER(T) cells. F ) Methylation index of GATA2 promoter CpG region 1 and 2 was determined using methylation specific primers designed using Methprimer. G ) PU/ER(T) cells were grown in presence of 95% Ethanol or 100 nM Tamoxifen for 24 h and total cellular RNA was isolated and reverse transcribed. Using the resulting cDNA as template relative expression levels of KLF4, GATA1 and GATA2 normalized to β-Actin were determined by gene specific primers and SYBR green reaction mix. Error bars represent standard deviation. Figure C & G * indicates p≤0.005 Tamoxifen treatment compared to control; Figure D & F * indicates p≤0.005 seven day differentiated BMDM compared to one day BMDM.
    Figure Legend Snippet: A ) Methprimer analysis of KLF4 promoter indicating dense CpG methylation distributed as three distinct regions, M1CpG proximal to the transcription start site and two distal M2 and M3CpG regions away from the transcription start site. B ) TF search analysis of 1.6 kb KLF4 promoter indicating putative PU.1 binding elements at −113 and −35 bp region from the transcription start site. C ) Total genomic DNA was isolated, Bisulfite converted and used as template for qPCR using methylation specific primers. The relative methylation index of M1, M2 and M3 CpG regions calculated as described in was compared in PUER(T) cells after treatment with Ethanol or Tamoxifen for 72 h and in D ) differentiating BMDM on 1 st and 7 th days E ) Methylation of M1CpG region was compared by bisulfite sequencing in control and 72 h 4-OHT treated PU/ER(T) cells. F ) Methylation index of GATA2 promoter CpG region 1 and 2 was determined using methylation specific primers designed using Methprimer. G ) PU/ER(T) cells were grown in presence of 95% Ethanol or 100 nM Tamoxifen for 24 h and total cellular RNA was isolated and reverse transcribed. Using the resulting cDNA as template relative expression levels of KLF4, GATA1 and GATA2 normalized to β-Actin were determined by gene specific primers and SYBR green reaction mix. Error bars represent standard deviation. Figure C & G * indicates p≤0.005 Tamoxifen treatment compared to control; Figure D & F * indicates p≤0.005 seven day differentiated BMDM compared to one day BMDM.

    Techniques Used: CpG Methylation Assay, Binding Assay, Isolation, Methylation, Methylation Sequencing, Expressing, SYBR Green Assay, Standard Deviation

    A ) PU/ER(T) cells were electroporated with 1.6 kb pGL3-KLF4 vector, after 16 h of electroporation treated with ethanol or 100 nM Tamoxifen for 24 h and analyzed for Luciferase reporter activity B ) HEK293 cells were transfected with empty pGL3, 1.6 kb KLF4-pGL3, pCMV-PU.1 or pCMV control vectors separately or in combination as indicated in figure, and after 36 h cell extracts were prepared in Promega cell lysis buffer and analyzed for luciferase activity. C ) HEK293 cells were transfected with empty pGL3, 1.6 kb mutant pGL3-KLF4, pCMV-PU.1 or pCMV control vectors separately or in combination as indicated in figure, and after 36 h cell extracts were prepared in Promega cell lysis buffer and analyzed for luciferase activity. D ) PU/ER(T) cells were treated with Tamoxifen or ethanol for indicated time periods and expression of KLF4 was determined by immunoblotting. E ) PU/ER(T) cells were electroporated with either control pCMV or KLF4 over expression vector and grown in IMDM for 72 h and analyzed for surface expression of F4/80. F ) The biotin labeled KLF4 promoter oligo was methylated using M.Sss I in presence of S-Adenosyl Methionine and used as a probe to determine the DNA binding activity in the Ethanol and Tamoxifen treated nuclear extracts as described in . G ) Empty pGL3 vector, pGL3-KLF4 Luciferase vector were in vitro methylated using M.SssI in presence of S-Adenosyl Methionine and electroporated along with control pCMV or pCMV-PU.1 expression vector. After 36 h, cell extracts were prepared in Promega cell lysis buffer and analyzed for luciferase activity. H ) Empty pcpgf-basic, pcpgf-KLF4 were in vitro methylated using M.sssI in presence of S-Adenosyl Methionine and electroporated along with control pCMV or pCMV-PU.1 expression vector in to HEK293 cells and grown in HEK293Blue detection medium for 36 h. Relative promoter activity was compared by mSEAP reporter activity as measured by the absorbance of the HEK293 Blue growth medium at 630 nm. For B, C, G, H reporter gene activities of pGL3-KLF4/pGL3-mutant KLF4/pcpg-KLF4 were not significantly different when co-transfected with the pCMV control plasmid. A to 4 C and 4 G, H means were compared and * is used where ever p value is ≤0.05 and ** is used when p≤0.005. Error bars represent standard deviation.
    Figure Legend Snippet: A ) PU/ER(T) cells were electroporated with 1.6 kb pGL3-KLF4 vector, after 16 h of electroporation treated with ethanol or 100 nM Tamoxifen for 24 h and analyzed for Luciferase reporter activity B ) HEK293 cells were transfected with empty pGL3, 1.6 kb KLF4-pGL3, pCMV-PU.1 or pCMV control vectors separately or in combination as indicated in figure, and after 36 h cell extracts were prepared in Promega cell lysis buffer and analyzed for luciferase activity. C ) HEK293 cells were transfected with empty pGL3, 1.6 kb mutant pGL3-KLF4, pCMV-PU.1 or pCMV control vectors separately or in combination as indicated in figure, and after 36 h cell extracts were prepared in Promega cell lysis buffer and analyzed for luciferase activity. D ) PU/ER(T) cells were treated with Tamoxifen or ethanol for indicated time periods and expression of KLF4 was determined by immunoblotting. E ) PU/ER(T) cells were electroporated with either control pCMV or KLF4 over expression vector and grown in IMDM for 72 h and analyzed for surface expression of F4/80. F ) The biotin labeled KLF4 promoter oligo was methylated using M.Sss I in presence of S-Adenosyl Methionine and used as a probe to determine the DNA binding activity in the Ethanol and Tamoxifen treated nuclear extracts as described in . G ) Empty pGL3 vector, pGL3-KLF4 Luciferase vector were in vitro methylated using M.SssI in presence of S-Adenosyl Methionine and electroporated along with control pCMV or pCMV-PU.1 expression vector. After 36 h, cell extracts were prepared in Promega cell lysis buffer and analyzed for luciferase activity. H ) Empty pcpgf-basic, pcpgf-KLF4 were in vitro methylated using M.sssI in presence of S-Adenosyl Methionine and electroporated along with control pCMV or pCMV-PU.1 expression vector in to HEK293 cells and grown in HEK293Blue detection medium for 36 h. Relative promoter activity was compared by mSEAP reporter activity as measured by the absorbance of the HEK293 Blue growth medium at 630 nm. For B, C, G, H reporter gene activities of pGL3-KLF4/pGL3-mutant KLF4/pcpg-KLF4 were not significantly different when co-transfected with the pCMV control plasmid. A to 4 C and 4 G, H means were compared and * is used where ever p value is ≤0.05 and ** is used when p≤0.005. Error bars represent standard deviation.

    Techniques Used: Plasmid Preparation, Electroporation, Luciferase, Activity Assay, Transfection, Lysis, Mutagenesis, Expressing, Western Blot, Over Expression, Labeling, Methylation, Binding Assay, In Vitro, Standard Deviation

    A ) PU/ER(T) cells were grown in presence of Ethanol or 100 nm Tamoxifen for the indicated time periods and cellular localization of AICDA, GADD45α, MBD4, TDG and TET2 was determined by immunoblotting with respective antibodies in cytosolic and nuclear protein fractions as described in . B ) AICDA was over expressed by electroporation of AICDA or Control plasmids in to PU/ER(T) cells and after 24 h of electroporation total AICDA and Myc in cell lysates was detected by immunoblotting. C ) Under similar conditions total AICDA in cell lysates was immunoprecipitated with anti myc Rabbit polyclonal antibodies and analyzed for co-immunoprecipitation of GADD45α, MBD4, TDG and TET2 by western blotting with respective antibodies. D ) Recruitment of AICDA to KLF4 promoter was assessed by ChIP in Ethanol or Tamoxifen treated PU/ER(T) cells and E ) differentiating bone derived marrow macrophages on Day 1, 2 and Day 7.
    Figure Legend Snippet: A ) PU/ER(T) cells were grown in presence of Ethanol or 100 nm Tamoxifen for the indicated time periods and cellular localization of AICDA, GADD45α, MBD4, TDG and TET2 was determined by immunoblotting with respective antibodies in cytosolic and nuclear protein fractions as described in . B ) AICDA was over expressed by electroporation of AICDA or Control plasmids in to PU/ER(T) cells and after 24 h of electroporation total AICDA and Myc in cell lysates was detected by immunoblotting. C ) Under similar conditions total AICDA in cell lysates was immunoprecipitated with anti myc Rabbit polyclonal antibodies and analyzed for co-immunoprecipitation of GADD45α, MBD4, TDG and TET2 by western blotting with respective antibodies. D ) Recruitment of AICDA to KLF4 promoter was assessed by ChIP in Ethanol or Tamoxifen treated PU/ER(T) cells and E ) differentiating bone derived marrow macrophages on Day 1, 2 and Day 7.

    Techniques Used: Western Blot, Electroporation, Immunoprecipitation, Derivative Assay

    A ) The relative methylation index of the proximal M1 CpG region in KLF4 promoter was compared on 1 st and 7 th day of differentiating BMDM from wild type and GADD45α knock out macrophages as described in C&D. B ) Knockdown of AICDA protein in AICDA-shRNA or control-shRNA electroporated PU/ER(T) cells. C ) Methylation index of the proximal KLF4 promoter M1 CpG region was compared in AICDA shRNA or control shRNA electroporated PU/ER(T) cells as described in . D ) Expression of F4/80 was analyzed in control shRNA or AICDA-shRNA electroporated PU/ER(T) cells treated with or without tamoxifen for 72 h. E ) Morphological changes in ethanol/tamoxifen treated PU/ER(T) cells were analyzed in AICDA depleted cells in comparison with control cells. Representative histograms of flow cytometry, western blots and cell staining were presented. , * is used where ever p value is ≤0.05 and ** is used when p≤0.005. Error bars represent standard deviation.
    Figure Legend Snippet: A ) The relative methylation index of the proximal M1 CpG region in KLF4 promoter was compared on 1 st and 7 th day of differentiating BMDM from wild type and GADD45α knock out macrophages as described in C&D. B ) Knockdown of AICDA protein in AICDA-shRNA or control-shRNA electroporated PU/ER(T) cells. C ) Methylation index of the proximal KLF4 promoter M1 CpG region was compared in AICDA shRNA or control shRNA electroporated PU/ER(T) cells as described in . D ) Expression of F4/80 was analyzed in control shRNA or AICDA-shRNA electroporated PU/ER(T) cells treated with or without tamoxifen for 72 h. E ) Morphological changes in ethanol/tamoxifen treated PU/ER(T) cells were analyzed in AICDA depleted cells in comparison with control cells. Representative histograms of flow cytometry, western blots and cell staining were presented. , * is used where ever p value is ≤0.05 and ** is used when p≤0.005. Error bars represent standard deviation.

    Techniques Used: Methylation, Knock-Out, shRNA, Expressing, Flow Cytometry, Western Blot, Staining, Standard Deviation



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    flag tagged human klf4 expression vectors  (OriGene)
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    OriGene flag tagged human klf4 expression vectors
    A ) PU/ER(T) cells were grown in presence of 95% Ethanol or 100 nM Tamoxifen for time periods as indicated. Cytosolic and nuclear protein fractions were prepared and translocation of PU.1 from cytosol to nucleus was detected by immunoblotting with anti PU.1 rabbit polyclonal antibodies. Purity of cytoplasmic and nuclear fractions was analyzed by immunoblotting with α-Tubulin and HDAC2 proteins. B ) Nuclear extracts were prepared from differentiating bone marrow derived macrophages on day 1 and 7 and in vitro binding to biotin labeled 76 bp oligonucleotide probe representing −118/−113 PU.1 binding element in the <t>KLF4</t> promoter was determined by EMSA as described in . C ) PU/ER(T) cells were treated with Ethanol or 100 nM Tamoxifen for 1, 4 or 24 h and nuclear extracts were prepared. An equal amount of nuclear protein extract from Ethanol and Tamoxifen treatment was analyzed for DNA binding activity using biotin labeled 76 bp oligonucleotide probe representing −118/−113 PU.1 binding element in the KLF4 promoter. Specificity of PU.1-KLF4 DNA complex in experiment B & C was determined by the ability of the PU.1-KLF4 DNA complex to form supershift with anti PU.1 antibody. D ) Specificity of PU.1-KLF4 DNA complex was determined by including 5, 15, 25 and 100 picomole of excess unlabelled KLF4 promoter oligo in the DNA binding reaction. E ) In vivo binding of PU.1 to KLF4 promoter was determined by Chromatin-immuno precipitation in differentiating BMDM on day 1, 2 and 7 and in F ) PU/ER(T) cells treated with Tamoxifen for 1 and 24 h. Error bars represent standard deviation and * is used where ever p value is ≤0.05 and ** is used when p≤0.005.
    Flag Tagged Human Klf4 Expression Vectors, supplied by OriGene, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/flag+tagged+human+klf4+expression+vectors/pmc03973678-46-4-13?v=OriGene
    Average 90 stars, based on 1 article reviews
    flag tagged human klf4 expression vectors - by Bioz Stars, 2026-08
    90/100 stars
    		  Buy from Supplier

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    A ) PU/ER(T) cells were grown in presence of 95% Ethanol or 100 nM Tamoxifen for time periods as indicated. Cytosolic and nuclear protein fractions were prepared and translocation of PU.1 from cytosol to nucleus was detected by immunoblotting with anti PU.1 rabbit polyclonal antibodies. Purity of cytoplasmic and nuclear fractions was analyzed by immunoblotting with α-Tubulin and HDAC2 proteins. B ) Nuclear extracts were prepared from differentiating bone marrow derived macrophages on day 1 and 7 and in vitro binding to biotin labeled 76 bp oligonucleotide probe representing −118/−113 PU.1 binding element in the KLF4 promoter was determined by EMSA as described in . C ) PU/ER(T) cells were treated with Ethanol or 100 nM Tamoxifen for 1, 4 or 24 h and nuclear extracts were prepared. An equal amount of nuclear protein extract from Ethanol and Tamoxifen treatment was analyzed for DNA binding activity using biotin labeled 76 bp oligonucleotide probe representing −118/−113 PU.1 binding element in the KLF4 promoter. Specificity of PU.1-KLF4 DNA complex in experiment B & C was determined by the ability of the PU.1-KLF4 DNA complex to form supershift with anti PU.1 antibody. D ) Specificity of PU.1-KLF4 DNA complex was determined by including 5, 15, 25 and 100 picomole of excess unlabelled KLF4 promoter oligo in the DNA binding reaction. E ) In vivo binding of PU.1 to KLF4 promoter was determined by Chromatin-immuno precipitation in differentiating BMDM on day 1, 2 and 7 and in F ) PU/ER(T) cells treated with Tamoxifen for 1 and 24 h. Error bars represent standard deviation and * is used where ever p value is ≤0.05 and ** is used when p≤0.005.

    Journal: PLoS ONE

    Article Title: Krüppel Like Factor 4 Promoter Undergoes Active Demethylation during Monocyte/Macrophage Differentiation

    doi: 10.1371/journal.pone.0093362

    Figure Lengend Snippet: A ) PU/ER(T) cells were grown in presence of 95% Ethanol or 100 nM Tamoxifen for time periods as indicated. Cytosolic and nuclear protein fractions were prepared and translocation of PU.1 from cytosol to nucleus was detected by immunoblotting with anti PU.1 rabbit polyclonal antibodies. Purity of cytoplasmic and nuclear fractions was analyzed by immunoblotting with α-Tubulin and HDAC2 proteins. B ) Nuclear extracts were prepared from differentiating bone marrow derived macrophages on day 1 and 7 and in vitro binding to biotin labeled 76 bp oligonucleotide probe representing −118/−113 PU.1 binding element in the KLF4 promoter was determined by EMSA as described in . C ) PU/ER(T) cells were treated with Ethanol or 100 nM Tamoxifen for 1, 4 or 24 h and nuclear extracts were prepared. An equal amount of nuclear protein extract from Ethanol and Tamoxifen treatment was analyzed for DNA binding activity using biotin labeled 76 bp oligonucleotide probe representing −118/−113 PU.1 binding element in the KLF4 promoter. Specificity of PU.1-KLF4 DNA complex in experiment B & C was determined by the ability of the PU.1-KLF4 DNA complex to form supershift with anti PU.1 antibody. D ) Specificity of PU.1-KLF4 DNA complex was determined by including 5, 15, 25 and 100 picomole of excess unlabelled KLF4 promoter oligo in the DNA binding reaction. E ) In vivo binding of PU.1 to KLF4 promoter was determined by Chromatin-immuno precipitation in differentiating BMDM on day 1, 2 and 7 and in F ) PU/ER(T) cells treated with Tamoxifen for 1 and 24 h. Error bars represent standard deviation and * is used where ever p value is ≤0.05 and ** is used when p≤0.005.

    Article Snippet: Myc tagged AICDA and FLAG tagged human KLF4 expression vectors were purchased from Origene (RC202949) and SABiosciences (DAM-603), respectively.

    Techniques: Translocation Assay, Western Blot, Derivative Assay, In Vitro, Binding Assay, Labeling, Activity Assay, In Vivo, Chromatin Immunoprecipitation, Standard Deviation

    A ) Methprimer analysis of KLF4 promoter indicating dense CpG methylation distributed as three distinct regions, M1CpG proximal to the transcription start site and two distal M2 and M3CpG regions away from the transcription start site. B ) TF search analysis of 1.6 kb KLF4 promoter indicating putative PU.1 binding elements at −113 and −35 bp region from the transcription start site. C ) Total genomic DNA was isolated, Bisulfite converted and used as template for qPCR using methylation specific primers. The relative methylation index of M1, M2 and M3 CpG regions calculated as described in was compared in PUER(T) cells after treatment with Ethanol or Tamoxifen for 72 h and in D ) differentiating BMDM on 1 st and 7 th days E ) Methylation of M1CpG region was compared by bisulfite sequencing in control and 72 h 4-OHT treated PU/ER(T) cells. F ) Methylation index of GATA2 promoter CpG region 1 and 2 was determined using methylation specific primers designed using Methprimer. G ) PU/ER(T) cells were grown in presence of 95% Ethanol or 100 nM Tamoxifen for 24 h and total cellular RNA was isolated and reverse transcribed. Using the resulting cDNA as template relative expression levels of KLF4, GATA1 and GATA2 normalized to β-Actin were determined by gene specific primers and SYBR green reaction mix. Error bars represent standard deviation. Figure C & G * indicates p≤0.005 Tamoxifen treatment compared to control; Figure D & F * indicates p≤0.005 seven day differentiated BMDM compared to one day BMDM.

    Journal: PLoS ONE

    Article Title: Krüppel Like Factor 4 Promoter Undergoes Active Demethylation during Monocyte/Macrophage Differentiation

    doi: 10.1371/journal.pone.0093362

    Figure Lengend Snippet: A ) Methprimer analysis of KLF4 promoter indicating dense CpG methylation distributed as three distinct regions, M1CpG proximal to the transcription start site and two distal M2 and M3CpG regions away from the transcription start site. B ) TF search analysis of 1.6 kb KLF4 promoter indicating putative PU.1 binding elements at −113 and −35 bp region from the transcription start site. C ) Total genomic DNA was isolated, Bisulfite converted and used as template for qPCR using methylation specific primers. The relative methylation index of M1, M2 and M3 CpG regions calculated as described in was compared in PUER(T) cells after treatment with Ethanol or Tamoxifen for 72 h and in D ) differentiating BMDM on 1 st and 7 th days E ) Methylation of M1CpG region was compared by bisulfite sequencing in control and 72 h 4-OHT treated PU/ER(T) cells. F ) Methylation index of GATA2 promoter CpG region 1 and 2 was determined using methylation specific primers designed using Methprimer. G ) PU/ER(T) cells were grown in presence of 95% Ethanol or 100 nM Tamoxifen for 24 h and total cellular RNA was isolated and reverse transcribed. Using the resulting cDNA as template relative expression levels of KLF4, GATA1 and GATA2 normalized to β-Actin were determined by gene specific primers and SYBR green reaction mix. Error bars represent standard deviation. Figure C & G * indicates p≤0.005 Tamoxifen treatment compared to control; Figure D & F * indicates p≤0.005 seven day differentiated BMDM compared to one day BMDM.

    Article Snippet: Myc tagged AICDA and FLAG tagged human KLF4 expression vectors were purchased from Origene (RC202949) and SABiosciences (DAM-603), respectively.

    Techniques: CpG Methylation Assay, Binding Assay, Isolation, Methylation, Methylation Sequencing, Expressing, SYBR Green Assay, Standard Deviation

    A ) PU/ER(T) cells were electroporated with 1.6 kb pGL3-KLF4 vector, after 16 h of electroporation treated with ethanol or 100 nM Tamoxifen for 24 h and analyzed for Luciferase reporter activity B ) HEK293 cells were transfected with empty pGL3, 1.6 kb KLF4-pGL3, pCMV-PU.1 or pCMV control vectors separately or in combination as indicated in figure, and after 36 h cell extracts were prepared in Promega cell lysis buffer and analyzed for luciferase activity. C ) HEK293 cells were transfected with empty pGL3, 1.6 kb mutant pGL3-KLF4, pCMV-PU.1 or pCMV control vectors separately or in combination as indicated in figure, and after 36 h cell extracts were prepared in Promega cell lysis buffer and analyzed for luciferase activity. D ) PU/ER(T) cells were treated with Tamoxifen or ethanol for indicated time periods and expression of KLF4 was determined by immunoblotting. E ) PU/ER(T) cells were electroporated with either control pCMV or KLF4 over expression vector and grown in IMDM for 72 h and analyzed for surface expression of F4/80. F ) The biotin labeled KLF4 promoter oligo was methylated using M.Sss I in presence of S-Adenosyl Methionine and used as a probe to determine the DNA binding activity in the Ethanol and Tamoxifen treated nuclear extracts as described in . G ) Empty pGL3 vector, pGL3-KLF4 Luciferase vector were in vitro methylated using M.SssI in presence of S-Adenosyl Methionine and electroporated along with control pCMV or pCMV-PU.1 expression vector. After 36 h, cell extracts were prepared in Promega cell lysis buffer and analyzed for luciferase activity. H ) Empty pcpgf-basic, pcpgf-KLF4 were in vitro methylated using M.sssI in presence of S-Adenosyl Methionine and electroporated along with control pCMV or pCMV-PU.1 expression vector in to HEK293 cells and grown in HEK293Blue detection medium for 36 h. Relative promoter activity was compared by mSEAP reporter activity as measured by the absorbance of the HEK293 Blue growth medium at 630 nm. For B, C, G, H reporter gene activities of pGL3-KLF4/pGL3-mutant KLF4/pcpg-KLF4 were not significantly different when co-transfected with the pCMV control plasmid. A to 4 C and 4 G, H means were compared and * is used where ever p value is ≤0.05 and ** is used when p≤0.005. Error bars represent standard deviation.

    Journal: PLoS ONE

    Article Title: Krüppel Like Factor 4 Promoter Undergoes Active Demethylation during Monocyte/Macrophage Differentiation

    doi: 10.1371/journal.pone.0093362

    Figure Lengend Snippet: A ) PU/ER(T) cells were electroporated with 1.6 kb pGL3-KLF4 vector, after 16 h of electroporation treated with ethanol or 100 nM Tamoxifen for 24 h and analyzed for Luciferase reporter activity B ) HEK293 cells were transfected with empty pGL3, 1.6 kb KLF4-pGL3, pCMV-PU.1 or pCMV control vectors separately or in combination as indicated in figure, and after 36 h cell extracts were prepared in Promega cell lysis buffer and analyzed for luciferase activity. C ) HEK293 cells were transfected with empty pGL3, 1.6 kb mutant pGL3-KLF4, pCMV-PU.1 or pCMV control vectors separately or in combination as indicated in figure, and after 36 h cell extracts were prepared in Promega cell lysis buffer and analyzed for luciferase activity. D ) PU/ER(T) cells were treated with Tamoxifen or ethanol for indicated time periods and expression of KLF4 was determined by immunoblotting. E ) PU/ER(T) cells were electroporated with either control pCMV or KLF4 over expression vector and grown in IMDM for 72 h and analyzed for surface expression of F4/80. F ) The biotin labeled KLF4 promoter oligo was methylated using M.Sss I in presence of S-Adenosyl Methionine and used as a probe to determine the DNA binding activity in the Ethanol and Tamoxifen treated nuclear extracts as described in . G ) Empty pGL3 vector, pGL3-KLF4 Luciferase vector were in vitro methylated using M.SssI in presence of S-Adenosyl Methionine and electroporated along with control pCMV or pCMV-PU.1 expression vector. After 36 h, cell extracts were prepared in Promega cell lysis buffer and analyzed for luciferase activity. H ) Empty pcpgf-basic, pcpgf-KLF4 were in vitro methylated using M.sssI in presence of S-Adenosyl Methionine and electroporated along with control pCMV or pCMV-PU.1 expression vector in to HEK293 cells and grown in HEK293Blue detection medium for 36 h. Relative promoter activity was compared by mSEAP reporter activity as measured by the absorbance of the HEK293 Blue growth medium at 630 nm. For B, C, G, H reporter gene activities of pGL3-KLF4/pGL3-mutant KLF4/pcpg-KLF4 were not significantly different when co-transfected with the pCMV control plasmid. A to 4 C and 4 G, H means were compared and * is used where ever p value is ≤0.05 and ** is used when p≤0.005. Error bars represent standard deviation.

    Article Snippet: Myc tagged AICDA and FLAG tagged human KLF4 expression vectors were purchased from Origene (RC202949) and SABiosciences (DAM-603), respectively.

    Techniques: Plasmid Preparation, Electroporation, Luciferase, Activity Assay, Transfection, Lysis, Mutagenesis, Expressing, Western Blot, Over Expression, Labeling, Methylation, Binding Assay, In Vitro, Standard Deviation

    A ) PU/ER(T) cells were grown in presence of Ethanol or 100 nm Tamoxifen for the indicated time periods and cellular localization of AICDA, GADD45α, MBD4, TDG and TET2 was determined by immunoblotting with respective antibodies in cytosolic and nuclear protein fractions as described in . B ) AICDA was over expressed by electroporation of AICDA or Control plasmids in to PU/ER(T) cells and after 24 h of electroporation total AICDA and Myc in cell lysates was detected by immunoblotting. C ) Under similar conditions total AICDA in cell lysates was immunoprecipitated with anti myc Rabbit polyclonal antibodies and analyzed for co-immunoprecipitation of GADD45α, MBD4, TDG and TET2 by western blotting with respective antibodies. D ) Recruitment of AICDA to KLF4 promoter was assessed by ChIP in Ethanol or Tamoxifen treated PU/ER(T) cells and E ) differentiating bone derived marrow macrophages on Day 1, 2 and Day 7.

    Journal: PLoS ONE

    Article Title: Krüppel Like Factor 4 Promoter Undergoes Active Demethylation during Monocyte/Macrophage Differentiation

    doi: 10.1371/journal.pone.0093362

    Figure Lengend Snippet: A ) PU/ER(T) cells were grown in presence of Ethanol or 100 nm Tamoxifen for the indicated time periods and cellular localization of AICDA, GADD45α, MBD4, TDG and TET2 was determined by immunoblotting with respective antibodies in cytosolic and nuclear protein fractions as described in . B ) AICDA was over expressed by electroporation of AICDA or Control plasmids in to PU/ER(T) cells and after 24 h of electroporation total AICDA and Myc in cell lysates was detected by immunoblotting. C ) Under similar conditions total AICDA in cell lysates was immunoprecipitated with anti myc Rabbit polyclonal antibodies and analyzed for co-immunoprecipitation of GADD45α, MBD4, TDG and TET2 by western blotting with respective antibodies. D ) Recruitment of AICDA to KLF4 promoter was assessed by ChIP in Ethanol or Tamoxifen treated PU/ER(T) cells and E ) differentiating bone derived marrow macrophages on Day 1, 2 and Day 7.

    Article Snippet: Myc tagged AICDA and FLAG tagged human KLF4 expression vectors were purchased from Origene (RC202949) and SABiosciences (DAM-603), respectively.

    Techniques: Western Blot, Electroporation, Immunoprecipitation, Derivative Assay

    A ) The relative methylation index of the proximal M1 CpG region in KLF4 promoter was compared on 1 st and 7 th day of differentiating BMDM from wild type and GADD45α knock out macrophages as described in C&D. B ) Knockdown of AICDA protein in AICDA-shRNA or control-shRNA electroporated PU/ER(T) cells. C ) Methylation index of the proximal KLF4 promoter M1 CpG region was compared in AICDA shRNA or control shRNA electroporated PU/ER(T) cells as described in . D ) Expression of F4/80 was analyzed in control shRNA or AICDA-shRNA electroporated PU/ER(T) cells treated with or without tamoxifen for 72 h. E ) Morphological changes in ethanol/tamoxifen treated PU/ER(T) cells were analyzed in AICDA depleted cells in comparison with control cells. Representative histograms of flow cytometry, western blots and cell staining were presented. , * is used where ever p value is ≤0.05 and ** is used when p≤0.005. Error bars represent standard deviation.

    Journal: PLoS ONE

    Article Title: Krüppel Like Factor 4 Promoter Undergoes Active Demethylation during Monocyte/Macrophage Differentiation

    doi: 10.1371/journal.pone.0093362

    Figure Lengend Snippet: A ) The relative methylation index of the proximal M1 CpG region in KLF4 promoter was compared on 1 st and 7 th day of differentiating BMDM from wild type and GADD45α knock out macrophages as described in C&D. B ) Knockdown of AICDA protein in AICDA-shRNA or control-shRNA electroporated PU/ER(T) cells. C ) Methylation index of the proximal KLF4 promoter M1 CpG region was compared in AICDA shRNA or control shRNA electroporated PU/ER(T) cells as described in . D ) Expression of F4/80 was analyzed in control shRNA or AICDA-shRNA electroporated PU/ER(T) cells treated with or without tamoxifen for 72 h. E ) Morphological changes in ethanol/tamoxifen treated PU/ER(T) cells were analyzed in AICDA depleted cells in comparison with control cells. Representative histograms of flow cytometry, western blots and cell staining were presented. , * is used where ever p value is ≤0.05 and ** is used when p≤0.005. Error bars represent standard deviation.

    Article Snippet: Myc tagged AICDA and FLAG tagged human KLF4 expression vectors were purchased from Origene (RC202949) and SABiosciences (DAM-603), respectively.

    Techniques: Methylation, Knock-Out, shRNA, Expressing, Flow Cytometry, Western Blot, Staining, Standard Deviation