tpa  (Millipore)

 
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    Name:
    SILuPrEST TPA
    Description:
    Lys Arg 13C and 15N metabolically labeled recombinant human protein fragment
    Catalog Number:
    QPREST22444
    Price:
    None
    Applications:
    Internal standard in MS-based quantitative proteomics
    Buy from Supplier


    Structured Review

    Millipore tpa
    Converting the -141 Zp nucleotide in the intact B95.8 genome to the Zp-V3 nucleotide increases lytic protein expression in stably infected Burkitt cells. (A) EBV-negative Mutu B cells were infected with wildtype, Zp mutant, or revertant B95.8 (2089) viruses as indicated, and stably selected with hygromycin B for two months. Two different independently selected lines for each virus were then treated for two days with or without <t>ionomycin</t> (in the presence or absence of cyclosporine), and immunoblots were performed to detect EBNA1, EBNA2, LMP1, Z, BMRF1 (early lytic protein), p18 (late lytic protein), and actin. Kem III cell extract was included as a positive control for EBNA1, EBNA2, and LMP1. (B) Mutu cell lines containing Wt or Zp mutant viruses were nucleofected with control siRNA or NFATc1 siRNA. Ionomycin or DMSO control was added after 48 hours, and cells harvested 72 hours post-infection. Immunoblots were performed to detect NFATc1, R, BMRF1, Z, and tubulin (loading control). (C) Mutu cell lines containing Wt or Zp mutant viruses (or mock infected cells) were treated with or without anti-IgG for two days and immunoblots performed to detect BMRF1, Z, and actin (loading control). (D) Mutu cell lines containing Wt, Zp mutant, or revertant viruses were treated with or without <t>TPA</t> plus sodium butyrate (NaBut) (in the presence or absence of cyclosporine) for two days and immunoblots performed to detect Z expression and GAPDH (loading control). (E) ChIP assays were performed using Mutu cell lines containing Wt or Zp mutant viruses treated for three hours with ionomycin. Formaldehyde-fixed cell extracts were immunoprecipitated with control anti-IgG or NFATc1 antibody. qPCR using primers for the EBV Z promoter was performed; results shown are expressed as the amount of Zp complexed to NFATc1 ab relative to the control IgG ab. Data represent three independent experiments.
    Lys Arg 13C and 15N metabolically labeled recombinant human protein fragment
    https://www.bioz.com/result/tpa/product/Millipore
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    tpa - by Bioz Stars, 2021-04
    93/100 stars

    Images

    1) Product Images from "A cancer-associated Epstein-Barr virus BZLF1 promoter variant enhances lytic infection"

    Article Title: A cancer-associated Epstein-Barr virus BZLF1 promoter variant enhances lytic infection

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007179

    Converting the -141 Zp nucleotide in the intact B95.8 genome to the Zp-V3 nucleotide increases lytic protein expression in stably infected Burkitt cells. (A) EBV-negative Mutu B cells were infected with wildtype, Zp mutant, or revertant B95.8 (2089) viruses as indicated, and stably selected with hygromycin B for two months. Two different independently selected lines for each virus were then treated for two days with or without ionomycin (in the presence or absence of cyclosporine), and immunoblots were performed to detect EBNA1, EBNA2, LMP1, Z, BMRF1 (early lytic protein), p18 (late lytic protein), and actin. Kem III cell extract was included as a positive control for EBNA1, EBNA2, and LMP1. (B) Mutu cell lines containing Wt or Zp mutant viruses were nucleofected with control siRNA or NFATc1 siRNA. Ionomycin or DMSO control was added after 48 hours, and cells harvested 72 hours post-infection. Immunoblots were performed to detect NFATc1, R, BMRF1, Z, and tubulin (loading control). (C) Mutu cell lines containing Wt or Zp mutant viruses (or mock infected cells) were treated with or without anti-IgG for two days and immunoblots performed to detect BMRF1, Z, and actin (loading control). (D) Mutu cell lines containing Wt, Zp mutant, or revertant viruses were treated with or without TPA plus sodium butyrate (NaBut) (in the presence or absence of cyclosporine) for two days and immunoblots performed to detect Z expression and GAPDH (loading control). (E) ChIP assays were performed using Mutu cell lines containing Wt or Zp mutant viruses treated for three hours with ionomycin. Formaldehyde-fixed cell extracts were immunoprecipitated with control anti-IgG or NFATc1 antibody. qPCR using primers for the EBV Z promoter was performed; results shown are expressed as the amount of Zp complexed to NFATc1 ab relative to the control IgG ab. Data represent three independent experiments.
    Figure Legend Snippet: Converting the -141 Zp nucleotide in the intact B95.8 genome to the Zp-V3 nucleotide increases lytic protein expression in stably infected Burkitt cells. (A) EBV-negative Mutu B cells were infected with wildtype, Zp mutant, or revertant B95.8 (2089) viruses as indicated, and stably selected with hygromycin B for two months. Two different independently selected lines for each virus were then treated for two days with or without ionomycin (in the presence or absence of cyclosporine), and immunoblots were performed to detect EBNA1, EBNA2, LMP1, Z, BMRF1 (early lytic protein), p18 (late lytic protein), and actin. Kem III cell extract was included as a positive control for EBNA1, EBNA2, and LMP1. (B) Mutu cell lines containing Wt or Zp mutant viruses were nucleofected with control siRNA or NFATc1 siRNA. Ionomycin or DMSO control was added after 48 hours, and cells harvested 72 hours post-infection. Immunoblots were performed to detect NFATc1, R, BMRF1, Z, and tubulin (loading control). (C) Mutu cell lines containing Wt or Zp mutant viruses (or mock infected cells) were treated with or without anti-IgG for two days and immunoblots performed to detect BMRF1, Z, and actin (loading control). (D) Mutu cell lines containing Wt, Zp mutant, or revertant viruses were treated with or without TPA plus sodium butyrate (NaBut) (in the presence or absence of cyclosporine) for two days and immunoblots performed to detect Z expression and GAPDH (loading control). (E) ChIP assays were performed using Mutu cell lines containing Wt or Zp mutant viruses treated for three hours with ionomycin. Formaldehyde-fixed cell extracts were immunoprecipitated with control anti-IgG or NFATc1 antibody. qPCR using primers for the EBV Z promoter was performed; results shown are expressed as the amount of Zp complexed to NFATc1 ab relative to the control IgG ab. Data represent three independent experiments.

    Techniques Used: Expressing, Stable Transfection, Infection, Mutagenesis, Western Blot, Positive Control, Chromatin Immunoprecipitation, Immunoprecipitation, Real-time Polymerase Chain Reaction

    2) Product Images from "Induction of apoptosis by TPA and VP-16 is through translocation of TR3"

    Article Title: Induction of apoptosis by TPA and VP-16 is through translocation of TR3

    Journal: World Journal of Gastroenterology

    doi: 10.3748/wjg.v8.i3.446

    Induction of apoptosis and TR3 expression induced by TPA and VP-16 in MGC80-3 cells. (A) Morphological analysis of apoptotic cells. Cells treated with TPA and VP-16 for 24 hr, and then stained with DAPI. Nuclear mor-phology was visualized under fluorescence microscope. (B) Measure of apoptotic index by counting 1000 cells stained with DAPI under fluorescence microscope. The data shown represents mean of three independent experiments (± SE). (C) Analysis of Bcl-2 protein expression. Cells were treated with TPA for indicated time, and Western blot was preformed as described in materials and methods. α-tubulin was used to quantify the amount of protein used in each lane. (D) Detection of TR3 mRNA expression. Cells were treated with TPA and VP-16 for 24 hr. Preparation of total RNA and Northern blot were carried out as described in materials and methods. 18S and 28S were shown to quantify the loading RNA. Lane 1: control; Lane 2: TPA treatment; Lane 3: VP-16 treatment.
    Figure Legend Snippet: Induction of apoptosis and TR3 expression induced by TPA and VP-16 in MGC80-3 cells. (A) Morphological analysis of apoptotic cells. Cells treated with TPA and VP-16 for 24 hr, and then stained with DAPI. Nuclear mor-phology was visualized under fluorescence microscope. (B) Measure of apoptotic index by counting 1000 cells stained with DAPI under fluorescence microscope. The data shown represents mean of three independent experiments (± SE). (C) Analysis of Bcl-2 protein expression. Cells were treated with TPA for indicated time, and Western blot was preformed as described in materials and methods. α-tubulin was used to quantify the amount of protein used in each lane. (D) Detection of TR3 mRNA expression. Cells were treated with TPA and VP-16 for 24 hr. Preparation of total RNA and Northern blot were carried out as described in materials and methods. 18S and 28S were shown to quantify the loading RNA. Lane 1: control; Lane 2: TPA treatment; Lane 3: VP-16 treatment.

    Techniques Used: Expressing, Staining, Fluorescence, Microscopy, Western Blot, Northern Blot

    3) Product Images from "Antibodies with Dual Reactivity to Plasminogen and Complementary PR3 in PR3-ANCA Vasculitis"

    Article Title: Antibodies with Dual Reactivity to Plasminogen and Complementary PR3 in PR3-ANCA Vasculitis

    Journal:

    doi: 10.1681/ASN.2008030270

    Functional effects of antiplasminogen antibodies. (A) An in vitro assay was performed to determine the rate of plasmin formation in the presence of antiplasminogen antibodies by combining plasminogen, uPA or tPA, and a chromogenic substrate with and without
    Figure Legend Snippet: Functional effects of antiplasminogen antibodies. (A) An in vitro assay was performed to determine the rate of plasmin formation in the presence of antiplasminogen antibodies by combining plasminogen, uPA or tPA, and a chromogenic substrate with and without

    Techniques Used: Functional Assay, In Vitro

    4) Product Images from "Deacetylase activity-independent transcriptional activation by HDAC2 during TPA-induced HL-60 cell differentiation"

    Article Title: Deacetylase activity-independent transcriptional activation by HDAC2 during TPA-induced HL-60 cell differentiation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0202935

    Recruitment of HDAC2 and PAX5 to the promoters of target genes. (A) Flag-tagged PAX5 was overexpressed in HEK293T cells. Extracts were immunoprecipitated with anti-Flag antibodies. Associated proteins were eluted, resolved by SDS-PAGE, and immunoblotted with anti-Flag and anti-HDAC2 antibodies. (B) Differential changes in interaction between HDAC2 and PAX5 48 hrs after treatment with 32 nM TPA were determined by IP. Extracts from TPA- or DMSO-treated HL-60 cells were immunoprecipitated with anti-PAX5 antibodies. HDAC2 that interacted with PAX5 in HL-60 cells was eluted and immunoblotted with a HDAC2 antibody. (C) The mRNA levels of atypically active genes in PAX5 -depleted HL-60 cells were determined by qPCR. PAX5 protein level was detected by western blotting. These data were normalized by GAPDH. (D) HEK293T cells were co-transfected with pCMV-Flag-PAX5 and pGL3.0- IL10RA or pGL3.0- RGCC promoters. Luciferase activities were measured 48 hrs after transfection, and normalized to that of β-galactosidase. (C–D) All results represent at least three independent experiments (± SEMs). * P
    Figure Legend Snippet: Recruitment of HDAC2 and PAX5 to the promoters of target genes. (A) Flag-tagged PAX5 was overexpressed in HEK293T cells. Extracts were immunoprecipitated with anti-Flag antibodies. Associated proteins were eluted, resolved by SDS-PAGE, and immunoblotted with anti-Flag and anti-HDAC2 antibodies. (B) Differential changes in interaction between HDAC2 and PAX5 48 hrs after treatment with 32 nM TPA were determined by IP. Extracts from TPA- or DMSO-treated HL-60 cells were immunoprecipitated with anti-PAX5 antibodies. HDAC2 that interacted with PAX5 in HL-60 cells was eluted and immunoblotted with a HDAC2 antibody. (C) The mRNA levels of atypically active genes in PAX5 -depleted HL-60 cells were determined by qPCR. PAX5 protein level was detected by western blotting. These data were normalized by GAPDH. (D) HEK293T cells were co-transfected with pCMV-Flag-PAX5 and pGL3.0- IL10RA or pGL3.0- RGCC promoters. Luciferase activities were measured 48 hrs after transfection, and normalized to that of β-galactosidase. (C–D) All results represent at least three independent experiments (± SEMs). * P

    Techniques Used: Immunoprecipitation, SDS Page, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Luciferase

    HDAC2 was recruited to the promoters of atypically active genes and induced transcription during HL-60 cell differentiation. (A–B) HL-60 cells were treated with TPA (32 nM) or DMSO for 48 hrs. (A) The occupancies of AcH3, H3K27me3, HDAC2, and RNA Pol II at the promoters of atypically active genes, typically active and repressive genes during HL-60 cell differentiation were analyzed. The data were normalized by input. These results are shown as means ± SDs (n = 3). (B) The differential changes in expression of the genes in TPA-treated HL-60 cells were confirmed by qPCR. (C) The mRNA levels of atypically active genes in HDAC2 -depleted HL-60 cells were analyzed using qPCR. Knockdown of HDAC2 was confirmed by western blotting. (B–C) These data were normalized by GAPDH. All results represent at least three independent experiments (± SEMs). * P
    Figure Legend Snippet: HDAC2 was recruited to the promoters of atypically active genes and induced transcription during HL-60 cell differentiation. (A–B) HL-60 cells were treated with TPA (32 nM) or DMSO for 48 hrs. (A) The occupancies of AcH3, H3K27me3, HDAC2, and RNA Pol II at the promoters of atypically active genes, typically active and repressive genes during HL-60 cell differentiation were analyzed. The data were normalized by input. These results are shown as means ± SDs (n = 3). (B) The differential changes in expression of the genes in TPA-treated HL-60 cells were confirmed by qPCR. (C) The mRNA levels of atypically active genes in HDAC2 -depleted HL-60 cells were analyzed using qPCR. Knockdown of HDAC2 was confirmed by western blotting. (B–C) These data were normalized by GAPDH. All results represent at least three independent experiments (± SEMs). * P

    Techniques Used: Cell Differentiation, Expressing, Real-time Polymerase Chain Reaction, Western Blot

    Genome-wide distribution profiles of AcH3, RNA Pol II, HDAC2, and H3K27me3 in TPA-treated HL-60 cells. (A) Heat map analysis showing AcH3, RNA Pol II, HDAC2, and H3K27me3 occupancies covering ± 3 kb region centered over each peak in DMSO- or TPA-treated HL-60 cells. The same color scales (white, no enrichment; red, high enrichment) were used for all data sets (upper panel). Merged profiles show each ChIP-seq signal around the TSS in DMSO- or TPA-treated HL-60 cells (lower panel) (B) Pie charts illustrate the genomic locations of AcH3, RNA Pol II, HDAC2, and H3K27me3 binding sites in TPA-treated HL-60 cells. (C) An area-proportional Venn diagram shows the overlap between AcH3, RNA Pol II, HDAC2, and H3K27me3 enrichment in TPA-treated HL-60 cells based on ChIP-seq data. (D) A heat map represents the binding profiles of AcH3, RNA Pol II, HDAC2, and H3K27me3 in TPA-treated HL-60 cells at the indicated target genes (± 3 kb around the TSSs). The same color scales (white, no enrichment; red, high enrichment) were used for all data sets. (E) ChIP-seq tracks of AcH3, RNA Pol II, HDAC2, and H3K27me3 in TPA-treated HL-60 cells along the AHNAK , USP13 , and SLC2A3 loci.
    Figure Legend Snippet: Genome-wide distribution profiles of AcH3, RNA Pol II, HDAC2, and H3K27me3 in TPA-treated HL-60 cells. (A) Heat map analysis showing AcH3, RNA Pol II, HDAC2, and H3K27me3 occupancies covering ± 3 kb region centered over each peak in DMSO- or TPA-treated HL-60 cells. The same color scales (white, no enrichment; red, high enrichment) were used for all data sets (upper panel). Merged profiles show each ChIP-seq signal around the TSS in DMSO- or TPA-treated HL-60 cells (lower panel) (B) Pie charts illustrate the genomic locations of AcH3, RNA Pol II, HDAC2, and H3K27me3 binding sites in TPA-treated HL-60 cells. (C) An area-proportional Venn diagram shows the overlap between AcH3, RNA Pol II, HDAC2, and H3K27me3 enrichment in TPA-treated HL-60 cells based on ChIP-seq data. (D) A heat map represents the binding profiles of AcH3, RNA Pol II, HDAC2, and H3K27me3 in TPA-treated HL-60 cells at the indicated target genes (± 3 kb around the TSSs). The same color scales (white, no enrichment; red, high enrichment) were used for all data sets. (E) ChIP-seq tracks of AcH3, RNA Pol II, HDAC2, and H3K27me3 in TPA-treated HL-60 cells along the AHNAK , USP13 , and SLC2A3 loci.

    Techniques Used: Genome Wide, Chromatin Immunoprecipitation, Binding Assay

    5) Product Images from "Positron emission tomography imaging of DMBA/TPA mouse skin multi-step tumorigenesis"

    Article Title: Positron emission tomography imaging of DMBA/TPA mouse skin multi-step tumorigenesis

    Journal: Molecular Oncology

    doi: 10.1016/j.molonc.2010.01.005

    Example longitudinal imaging of developing DMBA/TPA induced skin tumors. 18F‐FDG PET/CT images were taken at the indicated times after DMBA treatment (lower panels). To show the signal from skin tumor in two dimensional images, signals from internal organs are masked. Photos were taken at the same times (upper panels). Corresponding tumors are indicated by numbered arrows in each panel. The color scale indicates uptake relative to brain, with red being the highest, and lower values in yellow, green and blue. Signal intensities were normalized to the 18F‐FDG concentration in brain.
    Figure Legend Snippet: Example longitudinal imaging of developing DMBA/TPA induced skin tumors. 18F‐FDG PET/CT images were taken at the indicated times after DMBA treatment (lower panels). To show the signal from skin tumor in two dimensional images, signals from internal organs are masked. Photos were taken at the same times (upper panels). Corresponding tumors are indicated by numbered arrows in each panel. The color scale indicates uptake relative to brain, with red being the highest, and lower values in yellow, green and blue. Signal intensities were normalized to the 18F‐FDG concentration in brain.

    Techniques Used: Imaging, Positron Emission Tomography, Concentration Assay

    Representative histological views of DMBA/TPA induced skin tumor types. Tumors were harvested after the last imaging time points and processed for hematoxylin‐eosin staining. (A) Papilloma, grade 1–2. (B) Papilloma, grade 3. (C) Microinvasive squamous cell carcinoma, grade 1. (D) Microinvasive squamous cell carcinoma, grade 2. (No microinvasive squamous cell carcinomas, grade 3, were observed). (E) Fully invasive squamous cell carcinoma.
    Figure Legend Snippet: Representative histological views of DMBA/TPA induced skin tumor types. Tumors were harvested after the last imaging time points and processed for hematoxylin‐eosin staining. (A) Papilloma, grade 1–2. (B) Papilloma, grade 3. (C) Microinvasive squamous cell carcinoma, grade 1. (D) Microinvasive squamous cell carcinoma, grade 2. (No microinvasive squamous cell carcinomas, grade 3, were observed). (E) Fully invasive squamous cell carcinoma.

    Techniques Used: Imaging, Staining

    6) Product Images from "The transmembrane protein LRIG2 increases tumor progression in skin carcinogenesis"

    Article Title: The transmembrane protein LRIG2 increases tumor progression in skin carcinogenesis

    Journal: Molecular Oncology

    doi: 10.1002/1878-0261.12579

    TPA induces an increased inflammation of the skin of LRIG2‐TG mice. (A) Giemsa staining reveals huge inflammation spots and blood vessels (arrows) in the skin of LRIG2‐TG mice 48 h after a single application of TPA. Scale bars: 50 µm. (B) Morphometric measurements of the epidermal thickness showed less pronounced increase of epidermal thickness in LRIG2‐TG mice compared with controls after TPA treatment ( n = 4). (C) Western blot of phosphorylated and total ERBB receptors, MAPK1/2, PTEN, PCNA, CASP3, THBS1, and the inflammation markers: IL1A and IL6. GAPDH or TUBA1A were used as reference protein. (D) The proliferation index is not altered in LRIG2‐TG mice compared with controls ( n = 4). MKI67 staining of back skin of a TPA‐treated TG and control mouse. Scale bars: 50 µm. (E) Gelatin zymography with densitometric analysis of skin samples of TPA‐treated LRIG2‐TG mice and controls ( n = 4) revealed increased expression of pro‐MMP9. (F–I) Densitometric analysis of western blots in (C). Data are presented as mean + SEM and were analyzed by Student’s t ‐test. * P
    Figure Legend Snippet: TPA induces an increased inflammation of the skin of LRIG2‐TG mice. (A) Giemsa staining reveals huge inflammation spots and blood vessels (arrows) in the skin of LRIG2‐TG mice 48 h after a single application of TPA. Scale bars: 50 µm. (B) Morphometric measurements of the epidermal thickness showed less pronounced increase of epidermal thickness in LRIG2‐TG mice compared with controls after TPA treatment ( n = 4). (C) Western blot of phosphorylated and total ERBB receptors, MAPK1/2, PTEN, PCNA, CASP3, THBS1, and the inflammation markers: IL1A and IL6. GAPDH or TUBA1A were used as reference protein. (D) The proliferation index is not altered in LRIG2‐TG mice compared with controls ( n = 4). MKI67 staining of back skin of a TPA‐treated TG and control mouse. Scale bars: 50 µm. (E) Gelatin zymography with densitometric analysis of skin samples of TPA‐treated LRIG2‐TG mice and controls ( n = 4) revealed increased expression of pro‐MMP9. (F–I) Densitometric analysis of western blots in (C). Data are presented as mean + SEM and were analyzed by Student’s t ‐test. * P

    Techniques Used: Mouse Assay, Staining, Western Blot, Zymography, Expressing

    7) Product Images from "Keratinocyte p38δ loss inhibits Ras-induced tumor formation, while systemic p38δ loss enhances skin inflammation in the early phase of chemical carcinogenesis in mouse skin"

    Article Title: Keratinocyte p38δ loss inhibits Ras-induced tumor formation, while systemic p38δ loss enhances skin inflammation in the early phase of chemical carcinogenesis in mouse skin

    Journal: Molecular carcinogenesis

    doi: 10.1002/mc.22303

    Enhanced activation of p38α signaling in p38δ −/− skin in response to a short-term DMBA/TPA regimen, and in p38δ −/− keratinocytes in response to TNFα. ( A ) Total skin lysates were isolated from full-thickness dorsal skin of the individual p38δ +/+ and p38δ −/− mice 6 hours after the final TPA application, and expression of the indicated proteins was analyzed by immunoblot using the specified antibodies. p-p38, phosphorylated p38 MAPK. Pyk2 levels were assayed to assure equal protein loading. ( B ) Densitometric quantification of phosphorylated p38 bands. Data are normalized to the density of the total p38 bands (comprising p38α and p38δ bands in p38δ +/+ skin and p38α band only in p38δ −/− skin). * P
    Figure Legend Snippet: Enhanced activation of p38α signaling in p38δ −/− skin in response to a short-term DMBA/TPA regimen, and in p38δ −/− keratinocytes in response to TNFα. ( A ) Total skin lysates were isolated from full-thickness dorsal skin of the individual p38δ +/+ and p38δ −/− mice 6 hours after the final TPA application, and expression of the indicated proteins was analyzed by immunoblot using the specified antibodies. p-p38, phosphorylated p38 MAPK. Pyk2 levels were assayed to assure equal protein loading. ( B ) Densitometric quantification of phosphorylated p38 bands. Data are normalized to the density of the total p38 bands (comprising p38α and p38δ bands in p38δ +/+ skin and p38α band only in p38δ −/− skin). * P

    Techniques Used: Activation Assay, Isolation, Mouse Assay, Expressing

    Increased inflammatory cytokine expression and production, and exacerbated inflammation in p38δ −/− skin following a short-term DMBA/TPA challenge. Sets of p38δ +/+ and p38δ −/− . Total RNA ( A ) or total skin lysates ( B ) were isolated from full-thickness dorsal skin of mice (acetone vehicle control: n = 9; DMBA/TPA: n = 8–11 per genotype for RNA isolation; n = 10 per genotype for protein isolation) 2 hours after the final TPA application, and the levels of RNA or protein expression of the indicated mediators were analyzed using qRT-PCR ( A ) or ELISA ( B ), respectively. ( A ) qRT-RCR data from DMBA/TPA-treated skin are shown as fold changes over vehicle-treated skin and normalized to values from control p38δ +/+ mice. Results in ( A ) and ( B ) are shown as mean ± SE. * P
    Figure Legend Snippet: Increased inflammatory cytokine expression and production, and exacerbated inflammation in p38δ −/− skin following a short-term DMBA/TPA challenge. Sets of p38δ +/+ and p38δ −/− . Total RNA ( A ) or total skin lysates ( B ) were isolated from full-thickness dorsal skin of mice (acetone vehicle control: n = 9; DMBA/TPA: n = 8–11 per genotype for RNA isolation; n = 10 per genotype for protein isolation) 2 hours after the final TPA application, and the levels of RNA or protein expression of the indicated mediators were analyzed using qRT-PCR ( A ) or ELISA ( B ), respectively. ( A ) qRT-RCR data from DMBA/TPA-treated skin are shown as fold changes over vehicle-treated skin and normalized to values from control p38δ +/+ mice. Results in ( A ) and ( B ) are shown as mean ± SE. * P

    Techniques Used: Expressing, Isolation, Mouse Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    8) Product Images from "Early and sustained expression of latent and host modulating genes in coordinated transcriptional program of KSHV productive primary infection of human primary endothelial cells"

    Article Title: Early and sustained expression of latent and host modulating genes in coordinated transcriptional program of KSHV productive primary infection of human primary endothelial cells

    Journal: Virology

    doi: 10.1016/j.virol.2005.08.018

    Expression of KSHV transcripts in uninduced and TPA-induced BCBL-1 cells detected by reverse-transcription real-time quantitative PCR. (A) Scatter plot of C T values of uninduced ( y -axis) vs. TPA-induced ( x -axis) BCBL-1 cells. All C T values were the averages
    Figure Legend Snippet: Expression of KSHV transcripts in uninduced and TPA-induced BCBL-1 cells detected by reverse-transcription real-time quantitative PCR. (A) Scatter plot of C T values of uninduced ( y -axis) vs. TPA-induced ( x -axis) BCBL-1 cells. All C T values were the averages

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Related Articles

    Expressing:

    Article Title: Identification and Analysis of the K5 Gene of Kaposi's Sarcoma-Associated Herpesvirus
    Article Snippet: .. When necessary, lytic gene expression was induced in HHV-8-infected and control cells by treatment with TPA (Sigma) at 20 ng/μl. .. Cos-7 and 293T cells were maintained in Dulbecco modified Eagle medium supplemented with 10% FCS, penicillin, and streptomycin and were used for transient-transfection and cotransfection assays, respectively.

    ChIP-sequencing:

    Article Title: Gene-specific H1 eviction through a transcriptional activator-p300-NAP1-H1 pathway
    Article Snippet: The mRNA level for each expressed gene/transcript was analyzed by HOMER software ( ) with default parameters and represented as RPKM (reads per kilobase of transcript per million fragments mapped). .. ChIP-seq and ChIP-qPCR 697 or 697(FH-NAP1) cells were treated with 0.5 μg/ml TPA (Sigma) for 0-24 hrs. ..

    Chromatin Immunoprecipitation:

    Article Title: Gene-specific H1 eviction through a transcriptional activator-p300-NAP1-H1 pathway
    Article Snippet: The mRNA level for each expressed gene/transcript was analyzed by HOMER software ( ) with default parameters and represented as RPKM (reads per kilobase of transcript per million fragments mapped). .. ChIP-seq and ChIP-qPCR 697 or 697(FH-NAP1) cells were treated with 0.5 μg/ml TPA (Sigma) for 0-24 hrs. ..

    other:

    Article Title: Mechanisms of Chemical Cooperative Carcinogenesis by Epidermal Langerhans Cells
    Article Snippet: One week following initiation, twice-weekly applications of TPA (20 nmoles, Sigma) in ethanol began.

    Luciferase:

    Article Title: Sp1 and Sp3 regulate basal transcription of the human APOBEC3G gene
    Article Snippet: .. For stimulation of cells, final concentrations of 20 ng/ml TPA (Sigma) or 30 ng/ml IFN-α or 30 ng/ml IFN-γ (Tebu-Bio) were applied approximately 15 h before harvesting for luciferase assay. .. Electrophoretic mobility shift assay (EMSA) For preparation of nuclear extracts, 5 × 106 A3.01 T cells were washed in cold PBS and resuspended in 500 µl buffer A (10 mM HEPES pH7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF).

    Mouse Assay:

    Article Title: Pronounced cancer resistance in a subterranean rodent, the blind mole-rat, Spalax: in vivo and in vitro evidence
    Article Snippet: A single application of 200 μg of DMBA dissolved in 100 μl of acetone for mice, and 500 μg in 250 μl for Spalax was used. .. Three days after the initial DMBA dose, mice were treated with 30 μg of TPA (Sigma Aldrich, Inc.) dissolved in 100 μl of acetone, and Spalax with 60 μg of TPA dissolved in 250 μl of acetone. .. TPA was applied three times per week for two to three months, until all mice developed advanced cancer and were subsequently sacrificed.

    Concentration Assay:

    Article Title: Deacetylase activity-independent transcriptional activation by HDAC2 during TPA-induced HL-60 cell differentiation
    Article Snippet: .. For differentiation, HL-60 cells were seeded in a 100-mm plate at a concentration of 1 × 106 per mL and treated with 32 nM TPA (Sigma Aldrich) or DMSO (Duchefa). .. Plasmid constructs For the luciferase assay, the IL10RA and RGCC promoter regions (−998 to −1 and −1468 to +5, respectively) were amplified from genomic DNA using the primer pairs shown in and inserted into the pGL3.0-basic vector (Promega). pcDNA3-PAX5 was subcloned into the pCMV-Flag vector using primer pairs shown in .

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    Millipore tpa
    LC expression of CYP1B1 is required for optimal cutaneous chemical carcinogenesis ( a ) Chemical carcinogenesis was initiated in NLC and DTA controls as well as DTA repopulated with either w.t. or CYP1B1−/− LC using 400 <t>nmoles</t> DMBA followed by twice weekly <t>TPA</t> promotion (20 nmoles); ***P
    Tpa, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore tetradecanoyl phorbol acetate tpa
    Constitutive and inducible cellular kinases that phosphorylate the amino terminus of IκBα. S100 extract was prepared from HeLa cells that were either untreated (A) or treated with 50 ng of <t>phorbol</t> ester <t>(TPA)</t> per ml (B) for 1 h. The extract was fractionated on a phosphocellulose column, washed with 0.1 M KCl, and eluted with 0.3 M KCl. Each of these extracts was concentrated on a Q-Sepharose column and then fractionated on a Superdex 200 column. The column fractions from the Superdex 200 column were then assayed for their ability to bind and phosphorylate a GST-IκBα fusion protein truncated at amino acid 138. The positions of the molecular weight markers in the Superdex 200 column fractions are indicated. (C) Fraction 19 from untreated HeLa S100 extract that eluted from the mono Q column was assayed for its ability to phosphorylate 0.5 μg of either GST (lane 1), a GST-IκBα fusion protein truncated at amino acid 138 that contained wild-type or mutant sequences at serine residues 32 and 36 (lanes 2 and 3, respectively), and a GST-IκBα fusion protein truncated at amino acid 53 containing wild-type or mutant sequences at serine residues 32 and 36 (lanes 4 and 5, respectively). Similar results were obtained with TPA-treated extract.
    Tetradecanoyl Phorbol Acetate Tpa, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    LC expression of CYP1B1 is required for optimal cutaneous chemical carcinogenesis ( a ) Chemical carcinogenesis was initiated in NLC and DTA controls as well as DTA repopulated with either w.t. or CYP1B1−/− LC using 400 nmoles DMBA followed by twice weekly TPA promotion (20 nmoles); ***P

    Journal: The Journal of investigative dermatology

    Article Title: Mechanisms of Chemical Cooperative Carcinogenesis by Epidermal Langerhans Cells

    doi: 10.1038/jid.2014.411

    Figure Lengend Snippet: LC expression of CYP1B1 is required for optimal cutaneous chemical carcinogenesis ( a ) Chemical carcinogenesis was initiated in NLC and DTA controls as well as DTA repopulated with either w.t. or CYP1B1−/− LC using 400 nmoles DMBA followed by twice weekly TPA promotion (20 nmoles); ***P

    Article Snippet: One week following initiation, twice-weekly applications of TPA (20 nmoles, Sigma) in ethanol began.

    Techniques: Expressing

    LC facilitate DMBA-initiated cutaneous carcinogenesis and display phenotypic and morphologic changes in response to DMBA exposure ( a )Carcinogenesis initiated in LC-intact (NLC) and LC-deficient (DTA) mice by application of DMBA (400 nmoles) followed by twice weekly TPA (20 nmoles, n=13–15 mice/group, *** P ≤ 0.001). ( b ) XS106 was treated with 64μM DMBA and changes in gene expression monitored by quantitative real-time PCR, * P

    Journal: The Journal of investigative dermatology

    Article Title: Mechanisms of Chemical Cooperative Carcinogenesis by Epidermal Langerhans Cells

    doi: 10.1038/jid.2014.411

    Figure Lengend Snippet: LC facilitate DMBA-initiated cutaneous carcinogenesis and display phenotypic and morphologic changes in response to DMBA exposure ( a )Carcinogenesis initiated in LC-intact (NLC) and LC-deficient (DTA) mice by application of DMBA (400 nmoles) followed by twice weekly TPA (20 nmoles, n=13–15 mice/group, *** P ≤ 0.001). ( b ) XS106 was treated with 64μM DMBA and changes in gene expression monitored by quantitative real-time PCR, * P

    Article Snippet: One week following initiation, twice-weekly applications of TPA (20 nmoles, Sigma) in ethanol began.

    Techniques: Mouse Assay, Expressing, Real-time Polymerase Chain Reaction

    Linker histone H1 localization is correlated with gene expression during B cell differentiation. ). Values are represented as mean ± SD. (B) RT-qPCR analysis of selected genes without (grey bars) or with (red bars) TPA treatment. Signals were normalized to the HPRT mRNA level. n=4, mean±SD. (C) ChIP-qPCR for H1 on select genes without (grey bars) or with (red bars) TPA treatment. n=3, mean±SD. (D) Average of ChIP-qPCR results based on data in (C). mean±SD, *P

    Journal: Molecular cell

    Article Title: Gene-specific H1 eviction through a transcriptional activator-p300-NAP1-H1 pathway

    doi: 10.1016/j.molcel.2019.02.016

    Figure Lengend Snippet: Linker histone H1 localization is correlated with gene expression during B cell differentiation. ). Values are represented as mean ± SD. (B) RT-qPCR analysis of selected genes without (grey bars) or with (red bars) TPA treatment. Signals were normalized to the HPRT mRNA level. n=4, mean±SD. (C) ChIP-qPCR for H1 on select genes without (grey bars) or with (red bars) TPA treatment. n=3, mean±SD. (D) Average of ChIP-qPCR results based on data in (C). mean±SD, *P

    Article Snippet: ChIP-seq and ChIP-qPCR 697 or 697(FH-NAP1) cells were treated with 0.5 μg/ml TPA (Sigma) for 0-24 hrs.

    Techniques: Expressing, Cell Differentiation, Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    NAP1 depletion affects H1 loss and induction of the CD40 gene. 697 cells were either not transduced (no shRNA) or transduced with scramble (CTRL) or NAP1 shRNAs and treated without (gray bars) or with (red bars) TPA. (A) Knockdown of NAP1 mRNA. RT-qPCR values normalized to the no transfection/no TPA value. n=3, mean±SD. (B) CD40 (upper) and GAPDH (lower) mRNA levels n=3, mean±SD. (C-E) ChlP-qPCR assays on the CD40 .

    Journal: Molecular cell

    Article Title: Gene-specific H1 eviction through a transcriptional activator-p300-NAP1-H1 pathway

    doi: 10.1016/j.molcel.2019.02.016

    Figure Lengend Snippet: NAP1 depletion affects H1 loss and induction of the CD40 gene. 697 cells were either not transduced (no shRNA) or transduced with scramble (CTRL) or NAP1 shRNAs and treated without (gray bars) or with (red bars) TPA. (A) Knockdown of NAP1 mRNA. RT-qPCR values normalized to the no transfection/no TPA value. n=3, mean±SD. (B) CD40 (upper) and GAPDH (lower) mRNA levels n=3, mean±SD. (C-E) ChlP-qPCR assays on the CD40 .

    Article Snippet: ChIP-seq and ChIP-qPCR 697 or 697(FH-NAP1) cells were treated with 0.5 μg/ml TPA (Sigma) for 0-24 hrs.

    Techniques: shRNA, Transduction, Quantitative RT-PCR, Transfection, Real-time Polymerase Chain Reaction

    NAP1 moves from both sides of the CD40 promoter and expands the H1-free region following TPA-induced transcription. (A) H1 (upper panel) and H4ac (lower panel) occupancies on the CD40 locus. ChIP-qPCR in 697 cells. Primer positions (X-axis) and H1 and H4ac occupancies (Y-axis) are shown as symbols in gray (no TPA) or red (24 hr TPA). Gene positions are shown at the bottom and numbers are averages from two independent experiments. (B) Temporal spreading of H1 loss at the NCOA5-CD40 locus after TPA treatment. ChIP-qPCR for H1 with the X-axis indicating primer positions. (C) Temporal induction of NAP1 association and spreading at the CD40 locus after TPA treatment. ChIP-qPCR for FLAG-HA-NAP1 on CD40. X-axis indicates time after TPA and numbers are averages from two independent experiments. Primer sites (red arrows) and promoter NF-kB/SP1 sites on CD40 are shown at the top. (D) Gene expression at the CD40 locus after TPA treatment. Intergenic distances are shown at the top and mRNA levels (RT-qPCR) at the bottom. n=3, mean±SD. (E) H1 occupancy at the CD40 locus after TPA treatment. Results of ChIP-qPCR without (grey) or with (red) TPA. n=3, mean±SD. (F) CTCF binding sites at the CD40 locus. ChIP-seq with (lower) and without (upper) TPA treatment. (G) CTCF knockdown. mRNA levels (RT-qPCR) are shown for cells transduced with no, scrambled (CTRL) or CTCF shRNAs. (H) H1 loss at the CD40 locus after CTCF knockdown. ChIP-qPCR for cells transduced with scrambled (scr) or CTCF shRNAs with or without TPA treatment. Symbols indicate H1 values on the y-axis and primer positions on the x-axis relative to the NCOA5 and CD40 genes at the bottom. (I) CD40 .

    Journal: Molecular cell

    Article Title: Gene-specific H1 eviction through a transcriptional activator-p300-NAP1-H1 pathway

    doi: 10.1016/j.molcel.2019.02.016

    Figure Lengend Snippet: NAP1 moves from both sides of the CD40 promoter and expands the H1-free region following TPA-induced transcription. (A) H1 (upper panel) and H4ac (lower panel) occupancies on the CD40 locus. ChIP-qPCR in 697 cells. Primer positions (X-axis) and H1 and H4ac occupancies (Y-axis) are shown as symbols in gray (no TPA) or red (24 hr TPA). Gene positions are shown at the bottom and numbers are averages from two independent experiments. (B) Temporal spreading of H1 loss at the NCOA5-CD40 locus after TPA treatment. ChIP-qPCR for H1 with the X-axis indicating primer positions. (C) Temporal induction of NAP1 association and spreading at the CD40 locus after TPA treatment. ChIP-qPCR for FLAG-HA-NAP1 on CD40. X-axis indicates time after TPA and numbers are averages from two independent experiments. Primer sites (red arrows) and promoter NF-kB/SP1 sites on CD40 are shown at the top. (D) Gene expression at the CD40 locus after TPA treatment. Intergenic distances are shown at the top and mRNA levels (RT-qPCR) at the bottom. n=3, mean±SD. (E) H1 occupancy at the CD40 locus after TPA treatment. Results of ChIP-qPCR without (grey) or with (red) TPA. n=3, mean±SD. (F) CTCF binding sites at the CD40 locus. ChIP-seq with (lower) and without (upper) TPA treatment. (G) CTCF knockdown. mRNA levels (RT-qPCR) are shown for cells transduced with no, scrambled (CTRL) or CTCF shRNAs. (H) H1 loss at the CD40 locus after CTCF knockdown. ChIP-qPCR for cells transduced with scrambled (scr) or CTCF shRNAs with or without TPA treatment. Symbols indicate H1 values on the y-axis and primer positions on the x-axis relative to the NCOA5 and CD40 genes at the bottom. (I) CD40 .

    Article Snippet: ChIP-seq and ChIP-qPCR 697 or 697(FH-NAP1) cells were treated with 0.5 μg/ml TPA (Sigma) for 0-24 hrs.

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, Binding Assay, Transduction

    Recruitment of HDAC2 and PAX5 to the promoters of target genes. (A) Flag-tagged PAX5 was overexpressed in HEK293T cells. Extracts were immunoprecipitated with anti-Flag antibodies. Associated proteins were eluted, resolved by SDS-PAGE, and immunoblotted with anti-Flag and anti-HDAC2 antibodies. (B) Differential changes in interaction between HDAC2 and PAX5 48 hrs after treatment with 32 nM TPA were determined by IP. Extracts from TPA- or DMSO-treated HL-60 cells were immunoprecipitated with anti-PAX5 antibodies. HDAC2 that interacted with PAX5 in HL-60 cells was eluted and immunoblotted with a HDAC2 antibody. (C) The mRNA levels of atypically active genes in PAX5 -depleted HL-60 cells were determined by qPCR. PAX5 protein level was detected by western blotting. These data were normalized by GAPDH. (D) HEK293T cells were co-transfected with pCMV-Flag-PAX5 and pGL3.0- IL10RA or pGL3.0- RGCC promoters. Luciferase activities were measured 48 hrs after transfection, and normalized to that of β-galactosidase. (C–D) All results represent at least three independent experiments (± SEMs). * P

    Journal: PLoS ONE

    Article Title: Deacetylase activity-independent transcriptional activation by HDAC2 during TPA-induced HL-60 cell differentiation

    doi: 10.1371/journal.pone.0202935

    Figure Lengend Snippet: Recruitment of HDAC2 and PAX5 to the promoters of target genes. (A) Flag-tagged PAX5 was overexpressed in HEK293T cells. Extracts were immunoprecipitated with anti-Flag antibodies. Associated proteins were eluted, resolved by SDS-PAGE, and immunoblotted with anti-Flag and anti-HDAC2 antibodies. (B) Differential changes in interaction between HDAC2 and PAX5 48 hrs after treatment with 32 nM TPA were determined by IP. Extracts from TPA- or DMSO-treated HL-60 cells were immunoprecipitated with anti-PAX5 antibodies. HDAC2 that interacted with PAX5 in HL-60 cells was eluted and immunoblotted with a HDAC2 antibody. (C) The mRNA levels of atypically active genes in PAX5 -depleted HL-60 cells were determined by qPCR. PAX5 protein level was detected by western blotting. These data were normalized by GAPDH. (D) HEK293T cells were co-transfected with pCMV-Flag-PAX5 and pGL3.0- IL10RA or pGL3.0- RGCC promoters. Luciferase activities were measured 48 hrs after transfection, and normalized to that of β-galactosidase. (C–D) All results represent at least three independent experiments (± SEMs). * P

    Article Snippet: For differentiation, HL-60 cells were seeded in a 100-mm plate at a concentration of 1 × 106 per mL and treated with 32 nM TPA (Sigma Aldrich) or DMSO (Duchefa).

    Techniques: Immunoprecipitation, SDS Page, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Luciferase

    HDAC2 was recruited to the promoters of atypically active genes and induced transcription during HL-60 cell differentiation. (A–B) HL-60 cells were treated with TPA (32 nM) or DMSO for 48 hrs. (A) The occupancies of AcH3, H3K27me3, HDAC2, and RNA Pol II at the promoters of atypically active genes, typically active and repressive genes during HL-60 cell differentiation were analyzed. The data were normalized by input. These results are shown as means ± SDs (n = 3). (B) The differential changes in expression of the genes in TPA-treated HL-60 cells were confirmed by qPCR. (C) The mRNA levels of atypically active genes in HDAC2 -depleted HL-60 cells were analyzed using qPCR. Knockdown of HDAC2 was confirmed by western blotting. (B–C) These data were normalized by GAPDH. All results represent at least three independent experiments (± SEMs). * P

    Journal: PLoS ONE

    Article Title: Deacetylase activity-independent transcriptional activation by HDAC2 during TPA-induced HL-60 cell differentiation

    doi: 10.1371/journal.pone.0202935

    Figure Lengend Snippet: HDAC2 was recruited to the promoters of atypically active genes and induced transcription during HL-60 cell differentiation. (A–B) HL-60 cells were treated with TPA (32 nM) or DMSO for 48 hrs. (A) The occupancies of AcH3, H3K27me3, HDAC2, and RNA Pol II at the promoters of atypically active genes, typically active and repressive genes during HL-60 cell differentiation were analyzed. The data were normalized by input. These results are shown as means ± SDs (n = 3). (B) The differential changes in expression of the genes in TPA-treated HL-60 cells were confirmed by qPCR. (C) The mRNA levels of atypically active genes in HDAC2 -depleted HL-60 cells were analyzed using qPCR. Knockdown of HDAC2 was confirmed by western blotting. (B–C) These data were normalized by GAPDH. All results represent at least three independent experiments (± SEMs). * P

    Article Snippet: For differentiation, HL-60 cells were seeded in a 100-mm plate at a concentration of 1 × 106 per mL and treated with 32 nM TPA (Sigma Aldrich) or DMSO (Duchefa).

    Techniques: Cell Differentiation, Expressing, Real-time Polymerase Chain Reaction, Western Blot

    Genome-wide distribution profiles of AcH3, RNA Pol II, HDAC2, and H3K27me3 in TPA-treated HL-60 cells. (A) Heat map analysis showing AcH3, RNA Pol II, HDAC2, and H3K27me3 occupancies covering ± 3 kb region centered over each peak in DMSO- or TPA-treated HL-60 cells. The same color scales (white, no enrichment; red, high enrichment) were used for all data sets (upper panel). Merged profiles show each ChIP-seq signal around the TSS in DMSO- or TPA-treated HL-60 cells (lower panel) (B) Pie charts illustrate the genomic locations of AcH3, RNA Pol II, HDAC2, and H3K27me3 binding sites in TPA-treated HL-60 cells. (C) An area-proportional Venn diagram shows the overlap between AcH3, RNA Pol II, HDAC2, and H3K27me3 enrichment in TPA-treated HL-60 cells based on ChIP-seq data. (D) A heat map represents the binding profiles of AcH3, RNA Pol II, HDAC2, and H3K27me3 in TPA-treated HL-60 cells at the indicated target genes (± 3 kb around the TSSs). The same color scales (white, no enrichment; red, high enrichment) were used for all data sets. (E) ChIP-seq tracks of AcH3, RNA Pol II, HDAC2, and H3K27me3 in TPA-treated HL-60 cells along the AHNAK , USP13 , and SLC2A3 loci.

    Journal: PLoS ONE

    Article Title: Deacetylase activity-independent transcriptional activation by HDAC2 during TPA-induced HL-60 cell differentiation

    doi: 10.1371/journal.pone.0202935

    Figure Lengend Snippet: Genome-wide distribution profiles of AcH3, RNA Pol II, HDAC2, and H3K27me3 in TPA-treated HL-60 cells. (A) Heat map analysis showing AcH3, RNA Pol II, HDAC2, and H3K27me3 occupancies covering ± 3 kb region centered over each peak in DMSO- or TPA-treated HL-60 cells. The same color scales (white, no enrichment; red, high enrichment) were used for all data sets (upper panel). Merged profiles show each ChIP-seq signal around the TSS in DMSO- or TPA-treated HL-60 cells (lower panel) (B) Pie charts illustrate the genomic locations of AcH3, RNA Pol II, HDAC2, and H3K27me3 binding sites in TPA-treated HL-60 cells. (C) An area-proportional Venn diagram shows the overlap between AcH3, RNA Pol II, HDAC2, and H3K27me3 enrichment in TPA-treated HL-60 cells based on ChIP-seq data. (D) A heat map represents the binding profiles of AcH3, RNA Pol II, HDAC2, and H3K27me3 in TPA-treated HL-60 cells at the indicated target genes (± 3 kb around the TSSs). The same color scales (white, no enrichment; red, high enrichment) were used for all data sets. (E) ChIP-seq tracks of AcH3, RNA Pol II, HDAC2, and H3K27me3 in TPA-treated HL-60 cells along the AHNAK , USP13 , and SLC2A3 loci.

    Article Snippet: For differentiation, HL-60 cells were seeded in a 100-mm plate at a concentration of 1 × 106 per mL and treated with 32 nM TPA (Sigma Aldrich) or DMSO (Duchefa).

    Techniques: Genome Wide, Chromatin Immunoprecipitation, Binding Assay

    Constitutive and inducible cellular kinases that phosphorylate the amino terminus of IκBα. S100 extract was prepared from HeLa cells that were either untreated (A) or treated with 50 ng of phorbol ester (TPA) per ml (B) for 1 h. The extract was fractionated on a phosphocellulose column, washed with 0.1 M KCl, and eluted with 0.3 M KCl. Each of these extracts was concentrated on a Q-Sepharose column and then fractionated on a Superdex 200 column. The column fractions from the Superdex 200 column were then assayed for their ability to bind and phosphorylate a GST-IκBα fusion protein truncated at amino acid 138. The positions of the molecular weight markers in the Superdex 200 column fractions are indicated. (C) Fraction 19 from untreated HeLa S100 extract that eluted from the mono Q column was assayed for its ability to phosphorylate 0.5 μg of either GST (lane 1), a GST-IκBα fusion protein truncated at amino acid 138 that contained wild-type or mutant sequences at serine residues 32 and 36 (lanes 2 and 3, respectively), and a GST-IκBα fusion protein truncated at amino acid 53 containing wild-type or mutant sequences at serine residues 32 and 36 (lanes 4 and 5, respectively). Similar results were obtained with TPA-treated extract.

    Journal: Molecular and Cellular Biology

    Article Title: DNA-Dependent Protein Kinase Phosphorylation of I?B? and I?B? Regulates NF-?B DNA Binding Properties

    doi:

    Figure Lengend Snippet: Constitutive and inducible cellular kinases that phosphorylate the amino terminus of IκBα. S100 extract was prepared from HeLa cells that were either untreated (A) or treated with 50 ng of phorbol ester (TPA) per ml (B) for 1 h. The extract was fractionated on a phosphocellulose column, washed with 0.1 M KCl, and eluted with 0.3 M KCl. Each of these extracts was concentrated on a Q-Sepharose column and then fractionated on a Superdex 200 column. The column fractions from the Superdex 200 column were then assayed for their ability to bind and phosphorylate a GST-IκBα fusion protein truncated at amino acid 138. The positions of the molecular weight markers in the Superdex 200 column fractions are indicated. (C) Fraction 19 from untreated HeLa S100 extract that eluted from the mono Q column was assayed for its ability to phosphorylate 0.5 μg of either GST (lane 1), a GST-IκBα fusion protein truncated at amino acid 138 that contained wild-type or mutant sequences at serine residues 32 and 36 (lanes 2 and 3, respectively), and a GST-IκBα fusion protein truncated at amino acid 53 containing wild-type or mutant sequences at serine residues 32 and 36 (lanes 4 and 5, respectively). Similar results were obtained with TPA-treated extract.

    Article Snippet: Tetradecanoyl phorbol acetate (TPA) (Sigma; 50 ng/ml) and/or ionomycin (Calbiochem; 2 μM) was added to either HeLa, 3T3, or SF19 cells as outlined below.

    Techniques: Molecular Weight, Mutagenesis