Structured Review

TaKaRa erβ
<t>ERβ</t> isoforms (1, 2, and 5) are differentially expressed in melanoma cell lines. The relative expression of ERβ1, 2, and 5 isoforms was evaluated by quantitative RT-PCR, utilizing specific sets of primers. BLM and WM115 cells showed a similar expression of ERβ1 and 5, while expressing higher levels of ERβ2. On the other hand, a high expression of both ERβ2 and 5 isoforms (when compared to ERβ 1) was observed in A375 cells. One representative of three different experiments, which gave similar results, is shown.
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1) Product Images from "Estrogen Receptor β Agonists Differentially Affect the Growth of Human Melanoma Cell Lines"

Article Title: Estrogen Receptor β Agonists Differentially Affect the Growth of Human Melanoma Cell Lines

Journal: PLoS ONE

doi: 10.1371/journal.pone.0134396

ERβ isoforms (1, 2, and 5) are differentially expressed in melanoma cell lines. The relative expression of ERβ1, 2, and 5 isoforms was evaluated by quantitative RT-PCR, utilizing specific sets of primers. BLM and WM115 cells showed a similar expression of ERβ1 and 5, while expressing higher levels of ERβ2. On the other hand, a high expression of both ERβ2 and 5 isoforms (when compared to ERβ 1) was observed in A375 cells. One representative of three different experiments, which gave similar results, is shown.
Figure Legend Snippet: ERβ isoforms (1, 2, and 5) are differentially expressed in melanoma cell lines. The relative expression of ERβ1, 2, and 5 isoforms was evaluated by quantitative RT-PCR, utilizing specific sets of primers. BLM and WM115 cells showed a similar expression of ERβ1 and 5, while expressing higher levels of ERβ2. On the other hand, a high expression of both ERβ2 and 5 isoforms (when compared to ERβ 1) was observed in A375 cells. One representative of three different experiments, which gave similar results, is shown.

Techniques Used: Expressing, Quantitative RT-PCR

ERβ agonists significantly and specifically inhibit the proliferation of BLM melanoma cells. (A) BLM cells were treated with different doses of the classical ERβ agonist DPN every 48 h for three times. DPN significantly decreased cell proliferation at the dose of 10 −8 M. (B) Similar results were obtained when the cells were treated with E 2 . (C) The antiproliferative effect of both ERβ ligands (10 −8 M) was found to be specific since it was completely abrogated by cotreatment of the cells with the ER antagonist ICI 182,780 (10 −6 M). (D) BLM cells were treated with KB1, KB2, or KB4 (10 −9 , 10 −8 , 10 −7 M) every 48 h for three times. All three ERβ ligands significantly reduced cell proliferation at the dose of 10 −8 M. (E) BLM cells were treated with KB1, at different doses (10 9 −10 −7 M). The ERβ agonists decreased cell growth, being significantly effective at the dose of 10 −8 M. (F) The antiproliferative activity of KB1 (10 −8 M) was found to be specific since it was completely abrogated by cotreatment of the cells with the ER antagonist ICI 182,780. Each experimental group consisted of six replicates and each experiment was repeated three-five times. Results are given as cell number/plate. Data represent mean values ± SEM. * P
Figure Legend Snippet: ERβ agonists significantly and specifically inhibit the proliferation of BLM melanoma cells. (A) BLM cells were treated with different doses of the classical ERβ agonist DPN every 48 h for three times. DPN significantly decreased cell proliferation at the dose of 10 −8 M. (B) Similar results were obtained when the cells were treated with E 2 . (C) The antiproliferative effect of both ERβ ligands (10 −8 M) was found to be specific since it was completely abrogated by cotreatment of the cells with the ER antagonist ICI 182,780 (10 −6 M). (D) BLM cells were treated with KB1, KB2, or KB4 (10 −9 , 10 −8 , 10 −7 M) every 48 h for three times. All three ERβ ligands significantly reduced cell proliferation at the dose of 10 −8 M. (E) BLM cells were treated with KB1, at different doses (10 9 −10 −7 M). The ERβ agonists decreased cell growth, being significantly effective at the dose of 10 −8 M. (F) The antiproliferative activity of KB1 (10 −8 M) was found to be specific since it was completely abrogated by cotreatment of the cells with the ER antagonist ICI 182,780. Each experimental group consisted of six replicates and each experiment was repeated three-five times. Results are given as cell number/plate. Data represent mean values ± SEM. * P

Techniques Used: Activity Assay

ERβ, but not ERα, is expressed in human melanoma cells. (A) As a positive control, the expression of ERβ was evaluated by Western blot analysis in human BLM melanoma cells engineered to overexpress the receptor subtype protein, utilizing two primary antibodies: H-150 (Santa Cruz) and 14C8 (Abcam). A band corresponding to the receptor protein (59 kDa) was detected in basal conditions, both in control (C) and in Lipofectamine (Lipo) treated BLM cells. As expected, the intensity of this band was found to be significantly increased after ERβ overexpression (24–72 h), with the highest level of expression at 24 h. (B) By Western blot analysis, utilizing the two primary antibodies H-150 and 14C8, ERβ was found to be expressed at high levels in human BLM, A375, WM115, WM1552 melanoma cell lines (lanes 3, 4, 5, 7), while the human IGR-39 melanoma cell line expressed the receptor at almost undetectable levels (lane 6). ERβ was also expressed in human MCF-7 breast cancer cells, utilized as a positive control (lane 1), but it was not expressed in the human HEK293, utilized as a negative control. (C) On the other hand, all the human melanoma cells lines tested (lanes 2–6) did not express the ERα receptor isoform, which was expressed only in the control cell line (MCF-7, lane 1). β-actin was utilized as a loading control. For each analysis, one representative of three different experiments, which gave similar results, is shown.
Figure Legend Snippet: ERβ, but not ERα, is expressed in human melanoma cells. (A) As a positive control, the expression of ERβ was evaluated by Western blot analysis in human BLM melanoma cells engineered to overexpress the receptor subtype protein, utilizing two primary antibodies: H-150 (Santa Cruz) and 14C8 (Abcam). A band corresponding to the receptor protein (59 kDa) was detected in basal conditions, both in control (C) and in Lipofectamine (Lipo) treated BLM cells. As expected, the intensity of this band was found to be significantly increased after ERβ overexpression (24–72 h), with the highest level of expression at 24 h. (B) By Western blot analysis, utilizing the two primary antibodies H-150 and 14C8, ERβ was found to be expressed at high levels in human BLM, A375, WM115, WM1552 melanoma cell lines (lanes 3, 4, 5, 7), while the human IGR-39 melanoma cell line expressed the receptor at almost undetectable levels (lane 6). ERβ was also expressed in human MCF-7 breast cancer cells, utilized as a positive control (lane 1), but it was not expressed in the human HEK293, utilized as a negative control. (C) On the other hand, all the human melanoma cells lines tested (lanes 2–6) did not express the ERα receptor isoform, which was expressed only in the control cell line (MCF-7, lane 1). β-actin was utilized as a loading control. For each analysis, one representative of three different experiments, which gave similar results, is shown.

Techniques Used: Positive Control, Expressing, Western Blot, Over Expression, Negative Control

The specific ERβ ligand DPN affects the expression of cell cycle-related proteins in BLM melanoma cells. BLM cells were treated with DPN (10 −8 M) for 24, 48, or 72 h. Western blot analysis was performed on whole cell extracts by using specific antibodies against cell cycle-related proteins, such as cyclin D1, cyclin D3, p21, p27, CDK4, CDK6 (A) , procaspase-3 and cleaved caspase-3 (B) . Actin expression was evaluated as a loading control. The treatment with DPN reduced the expression of cyclin D1 and cyclin D3 and increased that of p27, while the levels of cleaved (active) caspase-3 were not affected by the treatment. One representative of three different experiments, which gave similar results, is shown. A statistical evaluation has been performed on the densitometric analysis of the results obtained from the three Western blot experiments performed on cell cycle-related proteins (A) .
Figure Legend Snippet: The specific ERβ ligand DPN affects the expression of cell cycle-related proteins in BLM melanoma cells. BLM cells were treated with DPN (10 −8 M) for 24, 48, or 72 h. Western blot analysis was performed on whole cell extracts by using specific antibodies against cell cycle-related proteins, such as cyclin D1, cyclin D3, p21, p27, CDK4, CDK6 (A) , procaspase-3 and cleaved caspase-3 (B) . Actin expression was evaluated as a loading control. The treatment with DPN reduced the expression of cyclin D1 and cyclin D3 and increased that of p27, while the levels of cleaved (active) caspase-3 were not affected by the treatment. One representative of three different experiments, which gave similar results, is shown. A statistical evaluation has been performed on the densitometric analysis of the results obtained from the three Western blot experiments performed on cell cycle-related proteins (A) .

Techniques Used: Expressing, Western Blot

ERβ activation induces global DNA methylation reprogramming in BLM melanoma cells. (A) Preliminary experiments were carried out to analyze the global DNA methylation status of BLM cells when compared to that of human normal melanocytes (hMel). To this purpose, a restriction enzymatic assay was employed. For each DNA sample, two restriction digests were performed: one with RsaI and MspI, and one with RsaI and HpaII. RsaI is methylation insensitive, while MspI and HpaII are sensitive to DNA methylation and are able to cut only unmethylated restriction sites. The digests were then amplified by PCR. Data are expressed as the MspI/RsaI or HpaII/RsaI ratios relative to the intensity of the bands. BLM melanoma cells were found to be globally hypomethylated when compared to normal melanocytes, when both MspI and HpaII restriction enzymes were utilized. One representative of three different experiments, which gave similar results, is reported. (B) Experiments were performed to evaluate whether activation of ERβ might affect the global DNA hypomethylation status observed in melanoma cells. BLM cells were treated with either DPN or E 2 (10 −8 M) for 24 or 48 h; the DNA methylation status was then evaluated as described above. Both DPN (at 24 and 48 h) and E 2 (at 24 h) increased the DNA methylation profile of BLM cells, indicating that ERβ activation reverts the DNA hypomethylation status in melanoma cells. One representative of three different experiments, which gave similar results, is reported.
Figure Legend Snippet: ERβ activation induces global DNA methylation reprogramming in BLM melanoma cells. (A) Preliminary experiments were carried out to analyze the global DNA methylation status of BLM cells when compared to that of human normal melanocytes (hMel). To this purpose, a restriction enzymatic assay was employed. For each DNA sample, two restriction digests were performed: one with RsaI and MspI, and one with RsaI and HpaII. RsaI is methylation insensitive, while MspI and HpaII are sensitive to DNA methylation and are able to cut only unmethylated restriction sites. The digests were then amplified by PCR. Data are expressed as the MspI/RsaI or HpaII/RsaI ratios relative to the intensity of the bands. BLM melanoma cells were found to be globally hypomethylated when compared to normal melanocytes, when both MspI and HpaII restriction enzymes were utilized. One representative of three different experiments, which gave similar results, is reported. (B) Experiments were performed to evaluate whether activation of ERβ might affect the global DNA hypomethylation status observed in melanoma cells. BLM cells were treated with either DPN or E 2 (10 −8 M) for 24 or 48 h; the DNA methylation status was then evaluated as described above. Both DPN (at 24 and 48 h) and E 2 (at 24 h) increased the DNA methylation profile of BLM cells, indicating that ERβ activation reverts the DNA hypomethylation status in melanoma cells. One representative of three different experiments, which gave similar results, is reported.

Techniques Used: Activation Assay, DNA Methylation Assay, Enzymatic Assay, Methylation, Amplification, Polymerase Chain Reaction

ERβ agonists differentially affect the proliferation of melanoma cell lines. (A) IGR-39, A375 and WM1552 melanoma cells were treated with DPN (10 −9 –10 −7 M) every 48 h for three times. No effect on cell proliferation could be observed in any cell line tested. (B) WM115 cells were treated with DPN, E 2 , or KB1 (10 −10 –10 −7 M) every 48 h for three times. DPN was significantly effective in decreasing cell proliferation at the doses of 10 −9 and 10 −8 M. On the other hand, both E 2 and KB1 significantly reduced cell proliferation at the dose of 10 −8 M. Each experimental group consisted of six replicates and each experiment was repeated three times. Results are given as cell number/plate. Data represent mean values ± SEM. * P
Figure Legend Snippet: ERβ agonists differentially affect the proliferation of melanoma cell lines. (A) IGR-39, A375 and WM1552 melanoma cells were treated with DPN (10 −9 –10 −7 M) every 48 h for three times. No effect on cell proliferation could be observed in any cell line tested. (B) WM115 cells were treated with DPN, E 2 , or KB1 (10 −10 –10 −7 M) every 48 h for three times. DPN was significantly effective in decreasing cell proliferation at the doses of 10 −9 and 10 −8 M. On the other hand, both E 2 and KB1 significantly reduced cell proliferation at the dose of 10 −8 M. Each experimental group consisted of six replicates and each experiment was repeated three times. Results are given as cell number/plate. Data represent mean values ± SEM. * P

Techniques Used:

ERβ ligands trigger cytoplasmic-to-nuclear translocation of ERβ and induce its transcriptional acitivity in BLM melanoma cells. (A) Immunofluorescence assay of ERβ intracellular localization. In control BLM melanoma cells, ERβ is mainly localized at the cytoplasmic level. Treatment of the cells with either DPN or E 2 (10 −8 M, for 24 h) induces ERβ translocation into the nucleus. A representative picture from three experiments done independently, which gave the same results, is reported. (B) The transcriptional activity of the ERβ protein in BLM cells was analyzed using the pVERE-tk-LUC plasmid (cotransfected with pCMVβ). The results were normalized for β-galactosidase activity. Treatment of the cells with either DPN or E 2 (10 −8 M, for 24 h) significantly increased ERβ transcriptional activity. Each experimental group consisted of three replicates and each experiment was repeated three times. Data represent mean values ± SEM. * P
Figure Legend Snippet: ERβ ligands trigger cytoplasmic-to-nuclear translocation of ERβ and induce its transcriptional acitivity in BLM melanoma cells. (A) Immunofluorescence assay of ERβ intracellular localization. In control BLM melanoma cells, ERβ is mainly localized at the cytoplasmic level. Treatment of the cells with either DPN or E 2 (10 −8 M, for 24 h) induces ERβ translocation into the nucleus. A representative picture from three experiments done independently, which gave the same results, is reported. (B) The transcriptional activity of the ERβ protein in BLM cells was analyzed using the pVERE-tk-LUC plasmid (cotransfected with pCMVβ). The results were normalized for β-galactosidase activity. Treatment of the cells with either DPN or E 2 (10 −8 M, for 24 h) significantly increased ERβ transcriptional activity. Each experimental group consisted of three replicates and each experiment was repeated three times. Data represent mean values ± SEM. * P

Techniques Used: Translocation Assay, Immunofluorescence, Activity Assay, Plasmid Preparation

2) Product Images from "Ets-1 as an early response gene against hypoxia-induced apoptosis in pancreatic β-cells"

Article Title: Ets-1 as an early response gene against hypoxia-induced apoptosis in pancreatic β-cells

Journal: Cell Death & Disease

doi: 10.1038/cddis.2015.8

Severe hypoxia-induced degradation of Ets-1 is correlated to its transactivation activity. ( a ) Effect of oxygen concentration on the protein level of Ets-1. MIN6 cells were exposed to 2, 4, 6, 8 and 10% O 2 for 1 h. Total proteins were extracted and analyzed by western blotting. ( b ) Effect of oxygen concentration on the transactivation activity of Ets-1. MIN6 cells were transfected with pEGFP-Ets-1 WT . At 24 h following transfection, cells were exposed to 2, 4, 6, 8 and 10% O 2 for 1 h, followed by RNA extraction and qRT-PCR analysis. The average values and standard deviations ( n =3) are shown. *and **indicate P
Figure Legend Snippet: Severe hypoxia-induced degradation of Ets-1 is correlated to its transactivation activity. ( a ) Effect of oxygen concentration on the protein level of Ets-1. MIN6 cells were exposed to 2, 4, 6, 8 and 10% O 2 for 1 h. Total proteins were extracted and analyzed by western blotting. ( b ) Effect of oxygen concentration on the transactivation activity of Ets-1. MIN6 cells were transfected with pEGFP-Ets-1 WT . At 24 h following transfection, cells were exposed to 2, 4, 6, 8 and 10% O 2 for 1 h, followed by RNA extraction and qRT-PCR analysis. The average values and standard deviations ( n =3) are shown. *and **indicate P

Techniques Used: Activity Assay, Concentration Assay, Western Blot, Transfection, RNA Extraction, Quantitative RT-PCR

Protective effect of Ets-1 on severe hypoxia-induced pancreatic β -cell apoptosis. ( a ) MIN6 cells were transiently transfected with pCMV5 (as a control) and pCMV5-Ets-1. At 24 h following transfection, the cells were exposed to 2% O 2 for 24 h, followed by annexin V-FITC/PI staining and flow cytometry analysis to determine the percentage of apoptotic cells. Dots in the lower right quadrant indicate annexin V + /PI − , early apoptotic cells, whereas dots in the upper right quadrant indicate annexin V + /PI + , late apoptotic cells. ( b ) Statistical graph of apoptotic cells as percentages. The Y -axis, (i.e., % apoptotic cells) indicates the percentage of early plus late apoptotic cells. The average values and standard deviations ( n =3) are shown. **indicates P
Figure Legend Snippet: Protective effect of Ets-1 on severe hypoxia-induced pancreatic β -cell apoptosis. ( a ) MIN6 cells were transiently transfected with pCMV5 (as a control) and pCMV5-Ets-1. At 24 h following transfection, the cells were exposed to 2% O 2 for 24 h, followed by annexin V-FITC/PI staining and flow cytometry analysis to determine the percentage of apoptotic cells. Dots in the lower right quadrant indicate annexin V + /PI − , early apoptotic cells, whereas dots in the upper right quadrant indicate annexin V + /PI + , late apoptotic cells. ( b ) Statistical graph of apoptotic cells as percentages. The Y -axis, (i.e., % apoptotic cells) indicates the percentage of early plus late apoptotic cells. The average values and standard deviations ( n =3) are shown. **indicates P

Techniques Used: Transfection, Staining, Flow Cytometry, Cytometry

Severe hypoxia promotes ubiquitin-proteasome-mediated degradation of Ets-1 protein in MIN6 cells. AMD, actinomycin D; H, hypoxia; N, normoxia. ( a ) Effect of severe hypoxia on pEGFP vector-mediated ectopic protein expression in MIN6 cells. MIN6 cells were transiently transfected with pEGFP (as a control) or pEGFP-Ets-1. Twenty-four hours after transfection, cells were maintained in normoxic condition or exposed to 2% O 2 for 1, 6 and 12 h, followed by total protein extraction and western blotting analysis. Anti-GFP antibody was used for immunoblotting of GFP and GFP-Ets-1 fusion protein. ( b ) Actinomycin D enhances hypoxia-induced decrease in Ets-1 protein level. After treatment with actinomycin D at the indicated concentrations, MIN6 cells were immediately exposed to 2% O 2 for 1 h followed by western blotting analysis. DMSO was used as the solvent control. ( c – e ) Ubiquitin-proteasome-mediated degradation of Ets-1 protein. ( c ) MIN6 cells were transiently transfected with pEGFP (as a control) or pEGFP-Ets-1. At 24 h following transfection, the cells were treated with MG132 at the indicated concentrations and then immediately exposed to 2% O 2 for 1 h. ( d ) MIN6 cells were treated with MG132 at the indicated concentrations and then immediately exposed to 2% O 2 for 1 h. ( c and d ) Total proteins were extracted and the protein levels of GFP-Ets-1 and endogenous Ets-1 were analyzed by western blotting using anti-Ets-1 antibody. ( a – d ) The left panels ( a – c ) and upper panel ( d ) show representative western blots. The right panels ( a – c ) and lower panel ( d ) show the relative quantification of normalized GFP, GFP-Ets-1 and Ets-1 levels to β -tubulin. The average values and standard deviations ( n =3) are shown. *and **indicate P
Figure Legend Snippet: Severe hypoxia promotes ubiquitin-proteasome-mediated degradation of Ets-1 protein in MIN6 cells. AMD, actinomycin D; H, hypoxia; N, normoxia. ( a ) Effect of severe hypoxia on pEGFP vector-mediated ectopic protein expression in MIN6 cells. MIN6 cells were transiently transfected with pEGFP (as a control) or pEGFP-Ets-1. Twenty-four hours after transfection, cells were maintained in normoxic condition or exposed to 2% O 2 for 1, 6 and 12 h, followed by total protein extraction and western blotting analysis. Anti-GFP antibody was used for immunoblotting of GFP and GFP-Ets-1 fusion protein. ( b ) Actinomycin D enhances hypoxia-induced decrease in Ets-1 protein level. After treatment with actinomycin D at the indicated concentrations, MIN6 cells were immediately exposed to 2% O 2 for 1 h followed by western blotting analysis. DMSO was used as the solvent control. ( c – e ) Ubiquitin-proteasome-mediated degradation of Ets-1 protein. ( c ) MIN6 cells were transiently transfected with pEGFP (as a control) or pEGFP-Ets-1. At 24 h following transfection, the cells were treated with MG132 at the indicated concentrations and then immediately exposed to 2% O 2 for 1 h. ( d ) MIN6 cells were treated with MG132 at the indicated concentrations and then immediately exposed to 2% O 2 for 1 h. ( c and d ) Total proteins were extracted and the protein levels of GFP-Ets-1 and endogenous Ets-1 were analyzed by western blotting using anti-Ets-1 antibody. ( a – d ) The left panels ( a – c ) and upper panel ( d ) show representative western blots. The right panels ( a – c ) and lower panel ( d ) show the relative quantification of normalized GFP, GFP-Ets-1 and Ets-1 levels to β -tubulin. The average values and standard deviations ( n =3) are shown. *and **indicate P

Techniques Used: Plasmid Preparation, Expressing, Transfection, Protein Extraction, Western Blot

Regulatory effect of Ets-1 on VEGFRs gene transcription and VEGFR3 luciferase reporter activity. ( a ) Relative quantification of GFP-Ets-1 protein. MIN6 cells were transfected with pEGFP-ETS-1 WT . At 24 h following transfection, cells were maintained in normoxic condition or exposed to 2% O 2 for 1 h, followed by western blotting analysis. The left panel shows a representative western blot. The right panel shows the relative quantification of normalized GFP-Ets-1 level to β -tubulin. The average values and standard deviations ( n =3) are shown. **indicates P
Figure Legend Snippet: Regulatory effect of Ets-1 on VEGFRs gene transcription and VEGFR3 luciferase reporter activity. ( a ) Relative quantification of GFP-Ets-1 protein. MIN6 cells were transfected with pEGFP-ETS-1 WT . At 24 h following transfection, cells were maintained in normoxic condition or exposed to 2% O 2 for 1 h, followed by western blotting analysis. The left panel shows a representative western blot. The right panel shows the relative quantification of normalized GFP-Ets-1 level to β -tubulin. The average values and standard deviations ( n =3) are shown. **indicates P

Techniques Used: Luciferase, Activity Assay, Transfection, Western Blot

Effects of hypoxia on Ets-1 mRNA and protein levels in MIN6 cells, primary cultured mouse/rat islets and α -TC6 cells. MIN6 cells ( a and b ), primary cultured mouse ( c and d )/rat ( e and f ) islets and α -TC6 cells ( g ) were exposed to 2% O 2 for 1–24 h. ( a , c and e ) Relative mRNA levels of Ets-1 were quantified by qRT-PCR analysis using β -actin as an internal control. The average values and standard deviations ( n =3) are shown. *and **indicate P
Figure Legend Snippet: Effects of hypoxia on Ets-1 mRNA and protein levels in MIN6 cells, primary cultured mouse/rat islets and α -TC6 cells. MIN6 cells ( a and b ), primary cultured mouse ( c and d )/rat ( e and f ) islets and α -TC6 cells ( g ) were exposed to 2% O 2 for 1–24 h. ( a , c and e ) Relative mRNA levels of Ets-1 were quantified by qRT-PCR analysis using β -actin as an internal control. The average values and standard deviations ( n =3) are shown. *and **indicate P

Techniques Used: Cell Culture, Quantitative RT-PCR

The pattern of multi-level regulation on Ets-1 during hypoxia. ( a ) Effect of moderate hypoxia on Ets-1 gene transcription in MIN6 cells. MIN6 cells were exposed to 10% O 2 for 6, 12, 16 and 24 h. Relative mRNA levels of Ets-1 were quantified by qRT-PCR analysis. The average values and standard deviations ( n =3) are shown. **indicates P
Figure Legend Snippet: The pattern of multi-level regulation on Ets-1 during hypoxia. ( a ) Effect of moderate hypoxia on Ets-1 gene transcription in MIN6 cells. MIN6 cells were exposed to 10% O 2 for 6, 12, 16 and 24 h. Relative mRNA levels of Ets-1 were quantified by qRT-PCR analysis. The average values and standard deviations ( n =3) are shown. **indicates P

Techniques Used: Quantitative RT-PCR

3) Product Images from "In Vivo Localization of Iris yellow spot Tospovirus (Bunyaviridae)-Encoded Proteins and Identification of Interacting Regions of Nucleocapsid and Movement Proteins"

Article Title: In Vivo Localization of Iris yellow spot Tospovirus (Bunyaviridae)-Encoded Proteins and Identification of Interacting Regions of Nucleocapsid and Movement Proteins

Journal: PLoS ONE

doi: 10.1371/journal.pone.0118973

Localization of Iris yellow spot virus (IYSV) proteins, in epidermal cells of IYSV-infected transgenic Nicotiana benthamiana plants containing the red nuclear marker histone 2B (RFP-H2B), and red endoplasmic reticulum marker (ER-RFP). Confocal micrographs represent IYSV fusion proteins to the C-terminus of green fluorescent protein (GFP). Columns from left to right show GFP-gene fusion (1), RFP-H2B (2), ER-RFP (3), and the overlay of the images (4). (a-d) GFP-IYSV N co-expression with RFP-H2B and ER-RFP; (e-h) GFP-IYSV NSm co-expression with RFP-H2B and ER-RFP; (i-l) GFP-IYSV NSs co-expression with RFP-H2B and ER-RFP; (m-p) GFP-IYSV G N co-expression with RFP-H2B and ER-RFP; (q-t) GFP-IYSV G C co-expression with RFP-H2B and ER-RFP. Each micrograph represents a minimum of 50 cells that were examined for localization. Scale bar = 20μm.
Figure Legend Snippet: Localization of Iris yellow spot virus (IYSV) proteins, in epidermal cells of IYSV-infected transgenic Nicotiana benthamiana plants containing the red nuclear marker histone 2B (RFP-H2B), and red endoplasmic reticulum marker (ER-RFP). Confocal micrographs represent IYSV fusion proteins to the C-terminus of green fluorescent protein (GFP). Columns from left to right show GFP-gene fusion (1), RFP-H2B (2), ER-RFP (3), and the overlay of the images (4). (a-d) GFP-IYSV N co-expression with RFP-H2B and ER-RFP; (e-h) GFP-IYSV NSm co-expression with RFP-H2B and ER-RFP; (i-l) GFP-IYSV NSs co-expression with RFP-H2B and ER-RFP; (m-p) GFP-IYSV G N co-expression with RFP-H2B and ER-RFP; (q-t) GFP-IYSV G C co-expression with RFP-H2B and ER-RFP. Each micrograph represents a minimum of 50 cells that were examined for localization. Scale bar = 20μm.

Techniques Used: Infection, Transgenic Assay, Marker, Expressing

Heterotypic interaction of Iris yellow spot virus (IYSV) nucleocapsid (N) and movement (NSm) proteins. (a) Schematic diagram of the constructs tested for leucine independent growth and β-galactosidase activity. Black box, LexA DBD in pEG202; hatched box, B42 TAD in pJG4-5; blue boxes, IYSV N (N1-full length, amino acids 1–273; N2, amino acids 1–90; N3, amino acids 91–180; N4, amino acids 181–220; N5, amino acids 221–273); yellow boxes, IYSV NSm (NSm1-full length, amino acids 1–312; NSm2, amino acids 1–160; NSm3, amino acids 100–200; NSm4, amino acids 201–312). Numbers to the left of each pair of constructions correspond to the β-galactosidase assays shown in (b). (b) β-galactosidase activity of yeast transformants expressing constructs as shown in (a).
Figure Legend Snippet: Heterotypic interaction of Iris yellow spot virus (IYSV) nucleocapsid (N) and movement (NSm) proteins. (a) Schematic diagram of the constructs tested for leucine independent growth and β-galactosidase activity. Black box, LexA DBD in pEG202; hatched box, B42 TAD in pJG4-5; blue boxes, IYSV N (N1-full length, amino acids 1–273; N2, amino acids 1–90; N3, amino acids 91–180; N4, amino acids 181–220; N5, amino acids 221–273); yellow boxes, IYSV NSm (NSm1-full length, amino acids 1–312; NSm2, amino acids 1–160; NSm3, amino acids 100–200; NSm4, amino acids 201–312). Numbers to the left of each pair of constructions correspond to the β-galactosidase assays shown in (b). (b) β-galactosidase activity of yeast transformants expressing constructs as shown in (a).

Techniques Used: Construct, Activity Assay, Expressing

Self-interaction of Iris yellow spot virus (IYSV) movement protein. (a) Schematic diagram of the constructs tested for leucine independent growth and β-galactosidase activity. Black box, LexA DBD in pEG202; hatched box, B42 TAD in pJG4-5; yellow boxes, IYSV NSm (NSm1-full length, amino acids 1–312; NSm2, amino acids 1–160; NSm3, amino acids 100–200; NSm4, amino acids 201–312); white box, full length Cauliflower mosaic virus P6 (amino acids 1–520). Numbers to the left of each pair of constructions correspond to the β-galactosidase assays shown in (b). (b) β-galactosidase activity of yeast transformants expressing constructs as shown in (a).
Figure Legend Snippet: Self-interaction of Iris yellow spot virus (IYSV) movement protein. (a) Schematic diagram of the constructs tested for leucine independent growth and β-galactosidase activity. Black box, LexA DBD in pEG202; hatched box, B42 TAD in pJG4-5; yellow boxes, IYSV NSm (NSm1-full length, amino acids 1–312; NSm2, amino acids 1–160; NSm3, amino acids 100–200; NSm4, amino acids 201–312); white box, full length Cauliflower mosaic virus P6 (amino acids 1–520). Numbers to the left of each pair of constructions correspond to the β-galactosidase assays shown in (b). (b) β-galactosidase activity of yeast transformants expressing constructs as shown in (a).

Techniques Used: Construct, Activity Assay, Expressing

In planta interactions of Iris yellow spot virus proteins as examined by bimolecular fluorescence complementation (BiFC) assay. Interaction assays were performed in leaf epidermal cells of IYSV-infected transgenic Nicotiana benthamiana plants expressing cyan fluorescent protein fused to the nuclear marker histone 2B (CFP-H2B), and cyan endoplasmic reticulum (ER-CFP) marker. Column 1 shows BiFC, column 2 shows localization of CFP-H2B and ER-CFP (nucleus and ER), and column 3 shows a merge of all panels (overlay). The first and second proteins mentioned in each pair of interactors were expressed as C-terminal fusions to the amino-terminal half of YFP and as C-terminal fusions to the carboxy-terminal half of YFP respectively. A set of positive interactions is shown here after testing interactions in all pairwise combinations: (a-c) N/N, (d-f) NSm/ NSm, (g-i) NSm/N. Each micrograph represents a minimum of 50 cells that were examined for interaction. Scale bar = 20μm.
Figure Legend Snippet: In planta interactions of Iris yellow spot virus proteins as examined by bimolecular fluorescence complementation (BiFC) assay. Interaction assays were performed in leaf epidermal cells of IYSV-infected transgenic Nicotiana benthamiana plants expressing cyan fluorescent protein fused to the nuclear marker histone 2B (CFP-H2B), and cyan endoplasmic reticulum (ER-CFP) marker. Column 1 shows BiFC, column 2 shows localization of CFP-H2B and ER-CFP (nucleus and ER), and column 3 shows a merge of all panels (overlay). The first and second proteins mentioned in each pair of interactors were expressed as C-terminal fusions to the amino-terminal half of YFP and as C-terminal fusions to the carboxy-terminal half of YFP respectively. A set of positive interactions is shown here after testing interactions in all pairwise combinations: (a-c) N/N, (d-f) NSm/ NSm, (g-i) NSm/N. Each micrograph represents a minimum of 50 cells that were examined for interaction. Scale bar = 20μm.

Techniques Used: Bimolecular Fluorescence Complementation Assay, Infection, Transgenic Assay, Expressing, Marker

Maltose binding protein pull-down assays of Iris yellow spot virus (IYSV) proteins. Self-interaction of nucleocapsid; N (1) and movement; NSm (2) proteins, and cross-interaction of IYSV N and NSm proteins (3). ‘Load’ above the column represents the protein content initially loaded to the MBP column; ‘wash’ above the column comprises of the protein not adhering to the column; ‘elution’ was the material that attached to the column and was desorbed by the addition of maltose. Proteins were subjected to protein gel blot analysis and probed with anti-GST primary antibody. (1.a) MBP-tagged N mixed with GST-tagged N. (1.b) MBP alone mixed with GST-tagged N. (1.c) MBP-tagged N mixed with GST alone. (1.d) GST alone mixed with MBP alone. (1.e) The same combination as 1.a, except that proteins were first subjected to RNase treatment. (2.a) MBP-tagged NSm mixed with GST-tagged NSm. (2.b) MBP alone mixed with GST-tagged NSm. (2.c) MBP-tagged NSm mixed with GST alone. (2.d) The same combination as 2.a, except that proteins were first subjected to RNase treatment. (3.a) MBP-tagged N mixed with GST-tagged NSm.
Figure Legend Snippet: Maltose binding protein pull-down assays of Iris yellow spot virus (IYSV) proteins. Self-interaction of nucleocapsid; N (1) and movement; NSm (2) proteins, and cross-interaction of IYSV N and NSm proteins (3). ‘Load’ above the column represents the protein content initially loaded to the MBP column; ‘wash’ above the column comprises of the protein not adhering to the column; ‘elution’ was the material that attached to the column and was desorbed by the addition of maltose. Proteins were subjected to protein gel blot analysis and probed with anti-GST primary antibody. (1.a) MBP-tagged N mixed with GST-tagged N. (1.b) MBP alone mixed with GST-tagged N. (1.c) MBP-tagged N mixed with GST alone. (1.d) GST alone mixed with MBP alone. (1.e) The same combination as 1.a, except that proteins were first subjected to RNase treatment. (2.a) MBP-tagged NSm mixed with GST-tagged NSm. (2.b) MBP alone mixed with GST-tagged NSm. (2.c) MBP-tagged NSm mixed with GST alone. (2.d) The same combination as 2.a, except that proteins were first subjected to RNase treatment. (3.a) MBP-tagged N mixed with GST-tagged NSm.

Techniques Used: Binding Assay, Western Blot

4) Product Images from "BnaA.bZIP1 Negatively Regulates a Novel Small Peptide Gene, BnaC.SP6, Involved in Pollen Activity"

Article Title: BnaA.bZIP1 Negatively Regulates a Novel Small Peptide Gene, BnaC.SP6, Involved in Pollen Activity

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2017.02117

BnaA.bZIP1 binds to the p158 promoter region and acts as a transcriptional repressor. (A) Growth of the co-transformed yeast cells on SD/-Leu plates without or with AbA (50 or 100 ng ml -1 ). (B) Diagram of the p158 region. The p158 region was divided into p103 and p99 regions, with a 43-bp overlap, containing putative C-box and ABRE motifs, respectively. The p59 and mp59 probes containing the C-box or mutated C-box were used for EMSA. The core sequence (CACGTC) of the C-box was changed to Ctatga (underlined). (C) Binding of BnaA.bZIP1 to the C-box of p158 region in EMSA. The BnaA.bZIP1-His protein was incubated with the Cy5-labeled probe containing the C-box or mutated C-box; the pET-32a-His protein was used as a negative control; the unlabeled promoter fragment was used as a competitor in the assay. -, absence; +, presence; black triangle indicates the shifted band; black arrows indicate reduced intensity of the shifted bands; a black star indicates the free probes. (D) Schematic representation of the constructs used for the dual luciferase reporter (DLR) assay in Arabidopsis protoplasts. BnaA.bZIP1 driven by the CaMV35S promoter was used as an effector. For each reporter construct, the firefly LUC gene was driven by the p158, p103, mutated p158 (mp158), and mutated p103 (mp103) promoter, respectively. (E) The promoter activity was indicated by a ratio of LUC to REN as described in Figure 4 ( n = 3). The value of the control was set to 1. Error bars represent the SE of three biological replicates.
Figure Legend Snippet: BnaA.bZIP1 binds to the p158 promoter region and acts as a transcriptional repressor. (A) Growth of the co-transformed yeast cells on SD/-Leu plates without or with AbA (50 or 100 ng ml -1 ). (B) Diagram of the p158 region. The p158 region was divided into p103 and p99 regions, with a 43-bp overlap, containing putative C-box and ABRE motifs, respectively. The p59 and mp59 probes containing the C-box or mutated C-box were used for EMSA. The core sequence (CACGTC) of the C-box was changed to Ctatga (underlined). (C) Binding of BnaA.bZIP1 to the C-box of p158 region in EMSA. The BnaA.bZIP1-His protein was incubated with the Cy5-labeled probe containing the C-box or mutated C-box; the pET-32a-His protein was used as a negative control; the unlabeled promoter fragment was used as a competitor in the assay. -, absence; +, presence; black triangle indicates the shifted band; black arrows indicate reduced intensity of the shifted bands; a black star indicates the free probes. (D) Schematic representation of the constructs used for the dual luciferase reporter (DLR) assay in Arabidopsis protoplasts. BnaA.bZIP1 driven by the CaMV35S promoter was used as an effector. For each reporter construct, the firefly LUC gene was driven by the p158, p103, mutated p158 (mp158), and mutated p103 (mp103) promoter, respectively. (E) The promoter activity was indicated by a ratio of LUC to REN as described in Figure 4 ( n = 3). The value of the control was set to 1. Error bars represent the SE of three biological replicates.

Techniques Used: Transformation Assay, Sequencing, Binding Assay, Incubation, Labeling, Positron Emission Tomography, Negative Control, Construct, Luciferase, Activity Assay

Expression pattern, subcellular localization, and the transcriptional activation activity of BnaA.bZIP1. (A) Expression levels of BnaA.bZIP1 in diverse tissues as assessed by qRT-PCR. Error bars indicate the SD and were calculated from three biological replicates. 3.5B, 0–3.5 mm buds; 6.5B, 3.5–6.5 mm buds; the other tissues are as mentioned in Figure 1C . (B) GUS expression in developing flower of pBnaA.bZIP1 transgenic lines. Bars, 500 μm (flower buds), 1 mm (whole inflorescence), and 100 μm in the case of stamen. (C) Subcellular localization of BnaA.bZIP1. The fusion plasmid (pM999-BnaA.bZIP1-GFP) and a nuclear marker plasmid (pM999-CFP-GHD7) were co-transformed into Arabidopsis protoplasts. Scale bars = 10 μm. (D) Assay for the transcriptional activation of BnaA.bZIP1 in yeast cells. The pGBKT7-AtbZIP1 and pGBKT7 were used as positive and negative controls, respectively. (E) Schematic diagrams of the constructs used for the transient expression assay in Arabidopsis protoplasts. The full-length and two C-terminal deletions of BnaA.bZIP1 were fused with GAL4BD. The pGAL4BD and pBD-AtbZIP1 were used as negative and positive controls, respectively. (F) The transcriptional activation abilities of effectors were determined by the ratio of LUC to REN, which was obtained from the co-transformation of protoplasts with the effector and reporter plasmids ( n = 3). The value of the negative control was set to 1. Error bars represent the SE of three biological replicates.
Figure Legend Snippet: Expression pattern, subcellular localization, and the transcriptional activation activity of BnaA.bZIP1. (A) Expression levels of BnaA.bZIP1 in diverse tissues as assessed by qRT-PCR. Error bars indicate the SD and were calculated from three biological replicates. 3.5B, 0–3.5 mm buds; 6.5B, 3.5–6.5 mm buds; the other tissues are as mentioned in Figure 1C . (B) GUS expression in developing flower of pBnaA.bZIP1 transgenic lines. Bars, 500 μm (flower buds), 1 mm (whole inflorescence), and 100 μm in the case of stamen. (C) Subcellular localization of BnaA.bZIP1. The fusion plasmid (pM999-BnaA.bZIP1-GFP) and a nuclear marker plasmid (pM999-CFP-GHD7) were co-transformed into Arabidopsis protoplasts. Scale bars = 10 μm. (D) Assay for the transcriptional activation of BnaA.bZIP1 in yeast cells. The pGBKT7-AtbZIP1 and pGBKT7 were used as positive and negative controls, respectively. (E) Schematic diagrams of the constructs used for the transient expression assay in Arabidopsis protoplasts. The full-length and two C-terminal deletions of BnaA.bZIP1 were fused with GAL4BD. The pGAL4BD and pBD-AtbZIP1 were used as negative and positive controls, respectively. (F) The transcriptional activation abilities of effectors were determined by the ratio of LUC to REN, which was obtained from the co-transformation of protoplasts with the effector and reporter plasmids ( n = 3). The value of the negative control was set to 1. Error bars represent the SE of three biological replicates.

Techniques Used: Expressing, Activation Assay, Activity Assay, Quantitative RT-PCR, Transgenic Assay, Plasmid Preparation, Marker, Transformation Assay, Construct, Negative Control

qRT-PCR analyses of genes bound and repressed by AtbZIP1 in DEX-induced BnaA.bZIP1/p158 plants. The data were calculated according to the 2 -ΔΔ C t method and the mRNA levels of genes on 0 day were set to 1. Bars show means ± SD ( n = 3). The RNA samples from buds were tested on 0, 4, and 6 days after treatment with DEX. (A–H) Relative mRNA levels of BnaA.bZIP1 , GUS ( Escherichia coli beta-glucuronidase gene), ATSP6 (At1g19500), LEA27 (LATE EMBRYOGENESIS ABUNDANT 27, At2g46140), GGP1 (GAMMA-GLUTAMYL PEPTIDASE 1, At4g30530), ATPS2 (PHOSPHATE STARVATION-INDUCED GENE 2, At1g73010), RIN4 (RPM1-interacting protein 4 family protein, At2g17660), and CML24 (CALMODULIN-LIKE 24, At5g37770).
Figure Legend Snippet: qRT-PCR analyses of genes bound and repressed by AtbZIP1 in DEX-induced BnaA.bZIP1/p158 plants. The data were calculated according to the 2 -ΔΔ C t method and the mRNA levels of genes on 0 day were set to 1. Bars show means ± SD ( n = 3). The RNA samples from buds were tested on 0, 4, and 6 days after treatment with DEX. (A–H) Relative mRNA levels of BnaA.bZIP1 , GUS ( Escherichia coli beta-glucuronidase gene), ATSP6 (At1g19500), LEA27 (LATE EMBRYOGENESIS ABUNDANT 27, At2g46140), GGP1 (GAMMA-GLUTAMYL PEPTIDASE 1, At4g30530), ATPS2 (PHOSPHATE STARVATION-INDUCED GENE 2, At1g73010), RIN4 (RPM1-interacting protein 4 family protein, At2g17660), and CML24 (CALMODULIN-LIKE 24, At5g37770).

Techniques Used: Quantitative RT-PCR

5) Product Images from "TGF-β induces p53/Smads complex formation in the PAI-1 promoter to activate transcription"

Article Title: TGF-β induces p53/Smads complex formation in the PAI-1 promoter to activate transcription

Journal: Scientific Reports

doi: 10.1038/srep35483

p53 selectively affects TGF-β target promoters containing both SBE and p53RE. ( a ) p53 did not significantly affect TGF-β-induced Smad7 promoter activation. HepG2 cells were transfected with Smad7 -Luc in the presence or absence of p53 expression plasmid. After 24 h, cells were treated with 100 pM of TGF-β. After 18 h, luciferase activity was measured. The experiments were performed in triplicate, and the data are represented as the mean-fold activation ± s.d. ( b ) A schematic representation of the human PAI-1 promoter constructs. ( c ) HepG2 cells were transfected with the indicated constructs. After 24 h, luciferase activity was measured as in ( a ). ( d ) HepG2 cells were transfected with the indicated constructs and siRNAs. After 24 h, luciferase activity was measured as in ( a ). ( e ) TGF-β could not transactivate a p53-responsive reporter. HepG2 cells were transfected with p53RE-Luc. After 24 h, cells were treated with 100 pM of TGF-β for 18 h. The luciferase activity was measured as in ( a ). ( f ) HaCaT cells were transiently transfected with the indicated siRNAs. After 48 h, expression of each gene was determined by semi-quantitative PCR. ( g ) HaCaT cells were transiently transfected with the indicated siRNAs. After 48 h, cells were treated with 100 pM of TGF-β for 6 h. The cell lysates were immunoblotted with the indicated antibodies. Uncropped images of gels/blots are shown in Supplementary Information, Figure S1 .
Figure Legend Snippet: p53 selectively affects TGF-β target promoters containing both SBE and p53RE. ( a ) p53 did not significantly affect TGF-β-induced Smad7 promoter activation. HepG2 cells were transfected with Smad7 -Luc in the presence or absence of p53 expression plasmid. After 24 h, cells were treated with 100 pM of TGF-β. After 18 h, luciferase activity was measured. The experiments were performed in triplicate, and the data are represented as the mean-fold activation ± s.d. ( b ) A schematic representation of the human PAI-1 promoter constructs. ( c ) HepG2 cells were transfected with the indicated constructs. After 24 h, luciferase activity was measured as in ( a ). ( d ) HepG2 cells were transfected with the indicated constructs and siRNAs. After 24 h, luciferase activity was measured as in ( a ). ( e ) TGF-β could not transactivate a p53-responsive reporter. HepG2 cells were transfected with p53RE-Luc. After 24 h, cells were treated with 100 pM of TGF-β for 18 h. The luciferase activity was measured as in ( a ). ( f ) HaCaT cells were transiently transfected with the indicated siRNAs. After 48 h, expression of each gene was determined by semi-quantitative PCR. ( g ) HaCaT cells were transiently transfected with the indicated siRNAs. After 48 h, cells were treated with 100 pM of TGF-β for 6 h. The cell lysates were immunoblotted with the indicated antibodies. Uncropped images of gels/blots are shown in Supplementary Information, Figure S1 .

Techniques Used: Activation Assay, Transfection, Expressing, Plasmid Preparation, Luciferase, Activity Assay, Construct, Real-time Polymerase Chain Reaction

6) Product Images from "Suppressive effects of RXR agonist PA024 on adrenal CYP11B2 expression, aldosterone secretion and blood pressure"

Article Title: Suppressive effects of RXR agonist PA024 on adrenal CYP11B2 expression, aldosterone secretion and blood pressure

Journal: PLoS ONE

doi: 10.1371/journal.pone.0181055

Effects of PA024 on the CYP11B2 promoter deletion mutants and point mutants. (A), effect of PA024 on the CYP11B2 promoter deletion mutants. Either -1521/+2-luc, -747/+2-luc, -135/+2-luc, -106/+2-luc, -65/+2-luc, or pGL3-Basic (control plasmid) was transiently transfected for 48 h with pCMV-β-gal into H295R cells, and the cells were thereafter treated with PA024 (10 μmol/L, 24 h) and Ang II (100 nmol/L, 6 h). Data represent mean ± S.E.M. (n = 4), percent of -1521/+2-luc control. (B), effect of PA024 on the CYP11B2 promoter point mutants. Either -1521/+2-luc, -1521/+2-luc-NBRE-1-mut, -1521/+2-luc-Ad5-mut, or -1521/+2-luc-Ad1/CRE-mut was transiently transfected for 48 h with pCMV-β-gal into H295R cells, and the cells were thereafter treated with PA024 (10 μmol/L, 24 h) and Ang II (100 nmol/L, 6 h). Data represent mean ± S.E.M. (n = 4), percent of -1521/+2-luc control. *** P
Figure Legend Snippet: Effects of PA024 on the CYP11B2 promoter deletion mutants and point mutants. (A), effect of PA024 on the CYP11B2 promoter deletion mutants. Either -1521/+2-luc, -747/+2-luc, -135/+2-luc, -106/+2-luc, -65/+2-luc, or pGL3-Basic (control plasmid) was transiently transfected for 48 h with pCMV-β-gal into H295R cells, and the cells were thereafter treated with PA024 (10 μmol/L, 24 h) and Ang II (100 nmol/L, 6 h). Data represent mean ± S.E.M. (n = 4), percent of -1521/+2-luc control. (B), effect of PA024 on the CYP11B2 promoter point mutants. Either -1521/+2-luc, -1521/+2-luc-NBRE-1-mut, -1521/+2-luc-Ad5-mut, or -1521/+2-luc-Ad1/CRE-mut was transiently transfected for 48 h with pCMV-β-gal into H295R cells, and the cells were thereafter treated with PA024 (10 μmol/L, 24 h) and Ang II (100 nmol/L, 6 h). Data represent mean ± S.E.M. (n = 4), percent of -1521/+2-luc control. *** P

Techniques Used: Plasmid Preparation, Transfection

7) Product Images from "p66? and p66? of the Mi-2/NuRD complex mediate MBD2 and histone interaction"

Article Title: p66? and p66? of the Mi-2/NuRD complex mediate MBD2 and histone interaction

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkj437

p66α- or p66β-mediated repression acts downstream of MBD2. Mouse fibroblasts ( 30 ) expressing wild-type MBD2 (MBD2+/+) ( A ) or MBD2 knock-out fibroblasts (MBD2−/−) ( B ) were transfected with vectors expressing the Gal-DNA binding domain, Gal-p66α or Gal-p66β together with a 4xUAStk luciferase reporter. Significant changes relative to Gal are indicated by asterisk.
Figure Legend Snippet: p66α- or p66β-mediated repression acts downstream of MBD2. Mouse fibroblasts ( 30 ) expressing wild-type MBD2 (MBD2+/+) ( A ) or MBD2 knock-out fibroblasts (MBD2−/−) ( B ) were transfected with vectors expressing the Gal-DNA binding domain, Gal-p66α or Gal-p66β together with a 4xUAStk luciferase reporter. Significant changes relative to Gal are indicated by asterisk.

Techniques Used: Expressing, Knock-Out, Transfection, Binding Assay, Luciferase

Knock-down of endogenous p66α or p66β decreases MBD2-mediated repression. ( A ) Endogenous expression of p66α and p66β is reduced 96 h after RNAi treatment. HeLa cells were transfected with either pSilencer constructs targeting p66α (pSil-αX), p66β (pSil-βX), a non-targeting control sequence (pSil-neg) or left untransfected (n.trans). Nuclear extracts were prepared and subjected to western blotting using the p66 antibody. ( B ) MBD2-mediated repression is reduced by knock-down of endogenous p66α or p66β. HeLa cells were transfected with the indicated pSilencer constructs together with expression vectors for the Gal-DNA binding domain or Gal-MBD2b and the 4xUAStk luciferase reporter. Cell extracts were analyzed for reporter gene activity. Fold repression was determined relative to the Gal-DNA binding domain. Error bars represent variations within duplicate transfections, significant changes relative to pSil-neg are indicated by asterisk.
Figure Legend Snippet: Knock-down of endogenous p66α or p66β decreases MBD2-mediated repression. ( A ) Endogenous expression of p66α and p66β is reduced 96 h after RNAi treatment. HeLa cells were transfected with either pSilencer constructs targeting p66α (pSil-αX), p66β (pSil-βX), a non-targeting control sequence (pSil-neg) or left untransfected (n.trans). Nuclear extracts were prepared and subjected to western blotting using the p66 antibody. ( B ) MBD2-mediated repression is reduced by knock-down of endogenous p66α or p66β. HeLa cells were transfected with the indicated pSilencer constructs together with expression vectors for the Gal-DNA binding domain or Gal-MBD2b and the 4xUAStk luciferase reporter. Cell extracts were analyzed for reporter gene activity. Fold repression was determined relative to the Gal-DNA binding domain. Error bars represent variations within duplicate transfections, significant changes relative to pSil-neg are indicated by asterisk.

Techniques Used: Expressing, Transfection, Construct, Sequencing, Western Blot, Binding Assay, Luciferase, Activity Assay

K149 of p66α is required for the MBD2 interaction as well as for the MBD2-mediated repression. ( A ) HEK293 cells were harvested 48 h after transfection with various combinations of DNA constructs, as indicated above the figure. Nuclear protein extracts were prepared (input) and purified with glutathione–Sepharose beads. The bound protein together with the input fractions were analyzed by western blotting using the anti-Gal antibody. ( B ) K149R mutant of p66α decreases MBD2-mediated repression. HeLa cells were cotransfected with a 4xUAStk luciferase reporter together with vectors expressing the Gal-DNA binding domain, or Gal-MBD2b and increasing amount of pSG5-p66α or pSG5-p66αK149R. Fold repression was determined relative to the Gal-DNA binding domain, significant changes relative to Gal-MBD2b (asterisk) and relative to comparable amounts of p66α (open triangle) are indicated.
Figure Legend Snippet: K149 of p66α is required for the MBD2 interaction as well as for the MBD2-mediated repression. ( A ) HEK293 cells were harvested 48 h after transfection with various combinations of DNA constructs, as indicated above the figure. Nuclear protein extracts were prepared (input) and purified with glutathione–Sepharose beads. The bound protein together with the input fractions were analyzed by western blotting using the anti-Gal antibody. ( B ) K149R mutant of p66α decreases MBD2-mediated repression. HeLa cells were cotransfected with a 4xUAStk luciferase reporter together with vectors expressing the Gal-DNA binding domain, or Gal-MBD2b and increasing amount of pSG5-p66α or pSG5-p66αK149R. Fold repression was determined relative to the Gal-DNA binding domain, significant changes relative to Gal-MBD2b (asterisk) and relative to comparable amounts of p66α (open triangle) are indicated.

Techniques Used: Transfection, Construct, Purification, Western Blot, Mutagenesis, Luciferase, Expressing, Binding Assay

Expression of p66α or p66β increases MBD2-mediated repression. HeLa cells were cotransfected with a 4xUAStk luciferase reporter together with plasmids coding for the Gal-DNA binding domain, Gal-MBD2b and no (mock) or increasing amounts of p66α ( A ) or p66β ( B ). Cell extracts were analyzed for reporter gene activity. Fold repression was determined relative to the Gal-DNA binding domain. Error bars represent variations within duplicate transfections, significant changes relative to mock are indicated by asterisk. Luciferase activity was measured 48 h after transfection.
Figure Legend Snippet: Expression of p66α or p66β increases MBD2-mediated repression. HeLa cells were cotransfected with a 4xUAStk luciferase reporter together with plasmids coding for the Gal-DNA binding domain, Gal-MBD2b and no (mock) or increasing amounts of p66α ( A ) or p66β ( B ). Cell extracts were analyzed for reporter gene activity. Fold repression was determined relative to the Gal-DNA binding domain. Error bars represent variations within duplicate transfections, significant changes relative to mock are indicated by asterisk. Luciferase activity was measured 48 h after transfection.

Techniques Used: Expressing, Luciferase, Binding Assay, Activity Assay, Transfection

8) Product Images from "Inhibition of Receptor Binding Stabilizes Newcastle Disease Virus HN and F Protein-Containing Complexes"

Article Title: Inhibition of Receptor Binding Stabilizes Newcastle Disease Virus HN and F Protein-Containing Complexes

Journal: Journal of Virology

doi: 10.1128/JVI.80.6.2894-2903.2006

HN protein receptor binding decreases amounts of HN and F protein complexes. (A) The fusion activities of mixtures of HN and F protein-expressing cells and target cells, either untreated or treated with neuraminidase (indicated at the bottom of the panel), were quantified using a β-galactosidase reporter assay. Cells cotransfected with HN and Fwt protein cDNAs as well as pTA were untreated or treated with the indicated concentrations of neuraminidase overnight. Target cells, transfected with pB1-G only, were untreated or incubated with the indicated concentrations of neuraminidase for 1 h prior to addition to HN and F protein-expressing cells. Fusion between the HN and F protein-expressing cells and target cells, quantified by measuring the β-galactosidase in cell extracts, is shown as the percent of activity detected in mixes of untreated cells. (B and C) Cells cotransfected with HN and F protein cDNAs were incubated overnight in 0.5 mg neuraminidase (+NA). Target cells were either untreated (−NA) (lanes 1 to 4) or treated with neuraminidase (+NA) for 1 h (lanes 5 to 8), washed, and then added on top of the HN and F protein-expressing cells, and the mix was incubated for 1 h at 37°C. Immune complexes were formed with antibodies (Ab) indicated at the top of panel B (IP Ab). IP, immunoprecipitation. The HN protein (electrophoresed in the presence of reducing agent) in the complexes was detected using anti-AS (IB HN), and the F protein (electrophoresed in the absence of reducing agent) was detected using anti-HR2 (IB F). IB, immunoblot. Anti-Fmix, a mixture of anti-HR1, anti-HR2, anti-F2-96, anti-Ftail, and anti-Fu1a. Anti-HNmAbs, a mixture of monoclonal antibodies. T, total cell extract, which represents 50% of the amount used for immunoprecipitation. (C) Bands in lanes 3 and 7 are a combination of comigrating F protein and precipitating rabbit antibody.
Figure Legend Snippet: HN protein receptor binding decreases amounts of HN and F protein complexes. (A) The fusion activities of mixtures of HN and F protein-expressing cells and target cells, either untreated or treated with neuraminidase (indicated at the bottom of the panel), were quantified using a β-galactosidase reporter assay. Cells cotransfected with HN and Fwt protein cDNAs as well as pTA were untreated or treated with the indicated concentrations of neuraminidase overnight. Target cells, transfected with pB1-G only, were untreated or incubated with the indicated concentrations of neuraminidase for 1 h prior to addition to HN and F protein-expressing cells. Fusion between the HN and F protein-expressing cells and target cells, quantified by measuring the β-galactosidase in cell extracts, is shown as the percent of activity detected in mixes of untreated cells. (B and C) Cells cotransfected with HN and F protein cDNAs were incubated overnight in 0.5 mg neuraminidase (+NA). Target cells were either untreated (−NA) (lanes 1 to 4) or treated with neuraminidase (+NA) for 1 h (lanes 5 to 8), washed, and then added on top of the HN and F protein-expressing cells, and the mix was incubated for 1 h at 37°C. Immune complexes were formed with antibodies (Ab) indicated at the top of panel B (IP Ab). IP, immunoprecipitation. The HN protein (electrophoresed in the presence of reducing agent) in the complexes was detected using anti-AS (IB HN), and the F protein (electrophoresed in the absence of reducing agent) was detected using anti-HR2 (IB F). IB, immunoblot. Anti-Fmix, a mixture of anti-HR1, anti-HR2, anti-F2-96, anti-Ftail, and anti-Fu1a. Anti-HNmAbs, a mixture of monoclonal antibodies. T, total cell extract, which represents 50% of the amount used for immunoprecipitation. (C) Bands in lanes 3 and 7 are a combination of comigrating F protein and precipitating rabbit antibody.

Techniques Used: Binding Assay, Expressing, Reporter Assay, Transfection, Incubation, Activity Assay, Immunoprecipitation

9) Product Images from "Identification of thioredoxin-interacting protein (TXNIP) as a downstream target for IGF1 action"

Article Title: Identification of thioredoxin-interacting protein (TXNIP) as a downstream target for IGF1 action

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1715930115

Transcriptional regulation of TXNIP promoter activity. ( A ) P69 cells were transiently transfected with a luciferase reporter under the control of the proximal TXNIP promoter (or empty pGL3 luciferase vector), along with a β-galactosidase vector. After 24 h, cells were cotransfected with an IGF1R expression vector (IGF1R-GFP), or empty vector (GFP), for an additional 24 h. At the end of the incubation, cells were harvested and luciferase and β-galactosidase values were measured. Promoter activities are expressed as luciferase normalized to β-galactosidase. ( B ) Autoregulation of the TXNIP gene. 3T3-L1 cells were transfected with a TXNIP promoter-luciferase gene (or empty pGL3), together with a β-galactosidase vector. Twenty-four hours after transfection, cells were cotransfected with a TXNIP expression vector (or empty GFP), and processed as described above. The results represent the mean ± SEM of three independent experiments. * P
Figure Legend Snippet: Transcriptional regulation of TXNIP promoter activity. ( A ) P69 cells were transiently transfected with a luciferase reporter under the control of the proximal TXNIP promoter (or empty pGL3 luciferase vector), along with a β-galactosidase vector. After 24 h, cells were cotransfected with an IGF1R expression vector (IGF1R-GFP), or empty vector (GFP), for an additional 24 h. At the end of the incubation, cells were harvested and luciferase and β-galactosidase values were measured. Promoter activities are expressed as luciferase normalized to β-galactosidase. ( B ) Autoregulation of the TXNIP gene. 3T3-L1 cells were transfected with a TXNIP promoter-luciferase gene (or empty pGL3), together with a β-galactosidase vector. Twenty-four hours after transfection, cells were cotransfected with a TXNIP expression vector (or empty GFP), and processed as described above. The results represent the mean ± SEM of three independent experiments. * P

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

10) Product Images from "Rolling Circle Translation of Circular RNA in Living Human Cells"

Article Title: Rolling Circle Translation of Circular RNA in Living Human Cells

Journal: Scientific Reports

doi: 10.1038/srep16435

Bicistronic reporter assay for IRES activity in the repeating FLAG-coding sequence. ( A ) Schematic representation of the bicistronic plasmid constructs. pβGal–CAT contains no insert between the two cistrons, which encoded chloramphenicol acetyltransferase (CAT) and β-galactosidase (β-gal). pβGal–4× FLAG–CAT contained a repeated (four) FLAG sequence and pβGal–IRES–CAT contained an IRES sequence derived from EMCV in the region between the two cistrons. The plasmid, pIRES, was used as a negative control. ( B,C ) Expression levels of β-gal ( B ) and CAT ( C ) in the cell lysate after the transfection of these plasmids into HeLa cells. The amounts of CAT and β-gal were determined by enzyme-linked immunoabsorbent assay. Results obtained from mock-transfected control are also shown. The plotted data are the means ± standard deviation of three independent experiments. ( D ) Relative IRES activities were calculated from the data shown in ( B , C ). The ratio of CAT/β-gal expression for pβGal–CAT was set at 1.0.
Figure Legend Snippet: Bicistronic reporter assay for IRES activity in the repeating FLAG-coding sequence. ( A ) Schematic representation of the bicistronic plasmid constructs. pβGal–CAT contains no insert between the two cistrons, which encoded chloramphenicol acetyltransferase (CAT) and β-galactosidase (β-gal). pβGal–4× FLAG–CAT contained a repeated (four) FLAG sequence and pβGal–IRES–CAT contained an IRES sequence derived from EMCV in the region between the two cistrons. The plasmid, pIRES, was used as a negative control. ( B,C ) Expression levels of β-gal ( B ) and CAT ( C ) in the cell lysate after the transfection of these plasmids into HeLa cells. The amounts of CAT and β-gal were determined by enzyme-linked immunoabsorbent assay. Results obtained from mock-transfected control are also shown. The plotted data are the means ± standard deviation of three independent experiments. ( D ) Relative IRES activities were calculated from the data shown in ( B , C ). The ratio of CAT/β-gal expression for pβGal–CAT was set at 1.0.

Techniques Used: Reporter Assay, Activity Assay, Sequencing, Plasmid Preparation, Construct, Derivative Assay, Negative Control, Expressing, Transfection, Standard Deviation

11) Product Images from "The proapoptotic function of Drosophila Hid is conserved in mammalian cells"

Article Title: The proapoptotic function of Drosophila Hid is conserved in mammalian cells

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

Hid localizes to the mitochondria. HeLa cells were transfected with pcDNA3 Hid. Twenty-four hours posttransfection, the cells were stained with 100 nM MitoTracker for 30 minutes, fixed, and immunostained with a mAb to Hid (CL1C3) and a FITC-conjugated secondary antibody. A – C shows a single HeLa cell visualized with indirect fluorescence microscopy filtered to allow FITC ( A ), MitoTracker ( B ), or both ( C ) to be seen. ( D ) Enlarged detail of the area indicated by the white box in C .
Figure Legend Snippet: Hid localizes to the mitochondria. HeLa cells were transfected with pcDNA3 Hid. Twenty-four hours posttransfection, the cells were stained with 100 nM MitoTracker for 30 minutes, fixed, and immunostained with a mAb to Hid (CL1C3) and a FITC-conjugated secondary antibody. A – C shows a single HeLa cell visualized with indirect fluorescence microscopy filtered to allow FITC ( A ), MitoTracker ( B ), or both ( C ) to be seen. ( D ) Enlarged detail of the area indicated by the white box in C .

Techniques Used: Transfection, Staining, Fluorescence, Microscopy

Hid induces apoptotic morphology in HeLa cells. HeLa cells were transiently transfected with 0.5 μg of pcDNA3 empty vector ( A ) or 0.5 μg of pcDNA3 Hid ( B ) along with 0.5 μg of pCMV-lacZ. Twenty-four hours posttransfection, the cells were fixed, stained with X-Gal, and observed by using light microscopy. ( C – E ) HeLa cells were transfected with 0.5 μg of pcDNA3 Hid along with 0.5 μg of pEGFP-CMV. At 24 hours, they were fixed, and the cells were stained with Hoechst dye no. 33358 (5 μg/ml) for 30 minutes, washed, and observed by using fluorescence microscopy on a FITC filter set ( C ) and a blue filterset ( D ). ( E ) Artificially merged images. ( F ) Quantification of apoptosis: The indicated cell types were transfected with 0.5 μg of pcDNA3 Hid or pcDNA3 along with a 0.5 μg of pCMV-lacZ. Twenty-four hours posttransfection, the cells were fixed and stained with X-Gal. Two hundred blue cells were counted in each of two duplicate transfections in three independent experiments. The fraction of blue stained cells with apoptotic morphology as a percentage of all blue-stained cells was calculated. The percentage is expressed as the mean ± SEM.
Figure Legend Snippet: Hid induces apoptotic morphology in HeLa cells. HeLa cells were transiently transfected with 0.5 μg of pcDNA3 empty vector ( A ) or 0.5 μg of pcDNA3 Hid ( B ) along with 0.5 μg of pCMV-lacZ. Twenty-four hours posttransfection, the cells were fixed, stained with X-Gal, and observed by using light microscopy. ( C – E ) HeLa cells were transfected with 0.5 μg of pcDNA3 Hid along with 0.5 μg of pEGFP-CMV. At 24 hours, they were fixed, and the cells were stained with Hoechst dye no. 33358 (5 μg/ml) for 30 minutes, washed, and observed by using fluorescence microscopy on a FITC filter set ( C ) and a blue filterset ( D ). ( E ) Artificially merged images. ( F ) Quantification of apoptosis: The indicated cell types were transfected with 0.5 μg of pcDNA3 Hid or pcDNA3 along with a 0.5 μg of pCMV-lacZ. Twenty-four hours posttransfection, the cells were fixed and stained with X-Gal. Two hundred blue cells were counted in each of two duplicate transfections in three independent experiments. The fraction of blue stained cells with apoptotic morphology as a percentage of all blue-stained cells was calculated. The percentage is expressed as the mean ± SEM.

Techniques Used: Transfection, Plasmid Preparation, Staining, Light Microscopy, Fluorescence, Microscopy

12) Product Images from "Group 13 HOX proteins interact with the MH2 domain of R-Smads and modulate Smad transcriptional activation functions independent of HOX DNA-binding capability"

Article Title: Group 13 HOX proteins interact with the MH2 domain of R-Smads and modulate Smad transcriptional activation functions independent of HOX DNA-binding capability

Journal: Nucleic Acids Research

doi: 10.1093/nar/gki761

HOX proteins repress Smad3-MH2 domain-mediated transcriptional activation. Hep3B cells in 12 well plates were co-transfected with pG5B-Luc reporter construct (500 ng), GAL4-Smad3C (left) or GAL4-VP16 (right) fusion protein construct (10 ng) with or without HOXA13 construct. β-galactosidase (40 ng) vector was included to correct for transfection efficiency. Experiments were carried out as described in the legend to Figure 3 , and luciferase activity in each sample was normalized to β-galactosidase activity; the average of two experiments is shown.
Figure Legend Snippet: HOX proteins repress Smad3-MH2 domain-mediated transcriptional activation. Hep3B cells in 12 well plates were co-transfected with pG5B-Luc reporter construct (500 ng), GAL4-Smad3C (left) or GAL4-VP16 (right) fusion protein construct (10 ng) with or without HOXA13 construct. β-galactosidase (40 ng) vector was included to correct for transfection efficiency. Experiments were carried out as described in the legend to Figure 3 , and luciferase activity in each sample was normalized to β-galactosidase activity; the average of two experiments is shown.

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

13) Product Images from "Pathogenicity of the BRCA1 missense variant M1775K is determined by the disruption of the BRCT phosphopeptide-binding pocket: a multi-modal approach"

Article Title: Pathogenicity of the BRCA1 missense variant M1775K is determined by the disruption of the BRCT phosphopeptide-binding pocket: a multi-modal approach

Journal:

doi: 10.1038/ejhg.2008.13

Functional analysis of M1775K in BRCA1. ( a ) Quantitative transcriptional assay in yeast cells. Cells were co-transformed with a LexA-responsive β -galactosidase reporter gene (diagram shown above the graph) and a LexA DBD fusion to residues 1396–1863
Figure Legend Snippet: Functional analysis of M1775K in BRCA1. ( a ) Quantitative transcriptional assay in yeast cells. Cells were co-transformed with a LexA-responsive β -galactosidase reporter gene (diagram shown above the graph) and a LexA DBD fusion to residues 1396–1863

Techniques Used: Functional Assay, Transcription Factor Assay, Transformation Assay

14) Product Images from "?-Adrenergic receptor-induced activation of nerve growth factor gene transcription in rat cerebral cortex involves CCAAT/enhancer-binding protein ?"

Article Title: ?-Adrenergic receptor-induced activation of nerve growth factor gene transcription in rat cerebral cortex involves CCAAT/enhancer-binding protein ?

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

Transactivation of NGF promoter by C/EBPδ. ( A Upper ) Schematic representation of the NGF promoter–luciferase construct. ( Lower ) Luciferase activity of the NGF promoter constructs in C6–2B cells. Luciferase activity was measured and normalized to the protein content and β-galactosidase activity. Data, expressed as percentage of the control (NGF−615/+50 with pMEX), are the mean ± SEM of 12 independent experiments, each performed in duplicate. ( B ) Luciferase activity of the NGF promoter constructs in PC12 cells. Cells were transfected and luciferase activity was measured as described above. Data are the mean ± SEM of 12 experiments, each performed in duplicate. ∗∗, P
Figure Legend Snippet: Transactivation of NGF promoter by C/EBPδ. ( A Upper ) Schematic representation of the NGF promoter–luciferase construct. ( Lower ) Luciferase activity of the NGF promoter constructs in C6–2B cells. Luciferase activity was measured and normalized to the protein content and β-galactosidase activity. Data, expressed as percentage of the control (NGF−615/+50 with pMEX), are the mean ± SEM of 12 independent experiments, each performed in duplicate. ( B ) Luciferase activity of the NGF promoter constructs in PC12 cells. Cells were transfected and luciferase activity was measured as described above. Data are the mean ± SEM of 12 experiments, each performed in duplicate. ∗∗, P

Techniques Used: Luciferase, Construct, Activity Assay, Transfection

Identification of the C/EBPδ-binding site in the NGF promoter. ( A ) DNase I footprinting analysis of the NGF promoter with recombinant C/EBPδ. The labeled fragment NGF−615+50 was incubated in the presence (+) or absence (−) of bacterially expressed C/EBPδ and partially digested with DNase I. Lanes G and A+G correspond to Maxam–Gilbert sequencing reactions performed on the same probe. Sequence of the protected area is shown. ( B ) Transactivation of 5′ deletion mutants of the NGF promoter by C/EBPδ in CTX-TNA2 cells. Each mutant was cotransfected with either pMEX or pMEX-C/EBPδ expression vectors and pRSV β-galactosidase reporter vector. The luciferase activity was measured 48 hr after transfection and normalized to β-galactosidase activity. Data, expressed as percentage of the full-length construct (NGF−615+50 with pMEX), are the mean ± SEM of three separate experiments, with two independent samples in each experiment. ∗, P
Figure Legend Snippet: Identification of the C/EBPδ-binding site in the NGF promoter. ( A ) DNase I footprinting analysis of the NGF promoter with recombinant C/EBPδ. The labeled fragment NGF−615+50 was incubated in the presence (+) or absence (−) of bacterially expressed C/EBPδ and partially digested with DNase I. Lanes G and A+G correspond to Maxam–Gilbert sequencing reactions performed on the same probe. Sequence of the protected area is shown. ( B ) Transactivation of 5′ deletion mutants of the NGF promoter by C/EBPδ in CTX-TNA2 cells. Each mutant was cotransfected with either pMEX or pMEX-C/EBPδ expression vectors and pRSV β-galactosidase reporter vector. The luciferase activity was measured 48 hr after transfection and normalized to β-galactosidase activity. Data, expressed as percentage of the full-length construct (NGF−615+50 with pMEX), are the mean ± SEM of three separate experiments, with two independent samples in each experiment. ∗, P

Techniques Used: Binding Assay, Footprinting, Recombinant, Labeling, Incubation, Sequencing, Mutagenesis, Expressing, Plasmid Preparation, Luciferase, Activity Assay, Transfection, Construct

15) Product Images from "Expressing functional siRNAs in mammalian cells using convergent transcription"

Article Title: Expressing functional siRNAs in mammalian cells using convergent transcription

Journal: BMC Biotechnology

doi: 10.1186/1472-6750-3-21

Suppression of p53 protein levels using a convergent U6 expression vector. (A) Transient suppression of p53 protein levels. Plasmids DualU6 and DualU6p53 or p53-specific siRNAs 1 and 2 were transfected into MDA MB 231 cells and, at 48 h and 120 h post-transfection, p53 and β-actin protein levels were determined using Western analysis. ( B ) Long term suppression of p53 protein levels. EcR293 cells containing a stably integrated dEGFP transgene were co-transfected with DualU6, DualU6GFP or DualU6p53 and pREP7. Following selection in hygromycin, cell populations were analysed for p53 and β-actin protein levels.
Figure Legend Snippet: Suppression of p53 protein levels using a convergent U6 expression vector. (A) Transient suppression of p53 protein levels. Plasmids DualU6 and DualU6p53 or p53-specific siRNAs 1 and 2 were transfected into MDA MB 231 cells and, at 48 h and 120 h post-transfection, p53 and β-actin protein levels were determined using Western analysis. ( B ) Long term suppression of p53 protein levels. EcR293 cells containing a stably integrated dEGFP transgene were co-transfected with DualU6, DualU6GFP or DualU6p53 and pREP7. Following selection in hygromycin, cell populations were analysed for p53 and β-actin protein levels.

Techniques Used: Expressing, Plasmid Preparation, Transfection, Multiple Displacement Amplification, Western Blot, Stable Transfection, Selection

16) Product Images from "Prostaglandin E Receptor Type 4-associated Protein Interacts Directly with NF-?B1 and Attenuates Macrophage Activation *Prostaglandin E Receptor Type 4-associated Protein Interacts Directly with NF-?B1 and Attenuates Macrophage Activation * S⃞"

Article Title: Prostaglandin E Receptor Type 4-associated Protein Interacts Directly with NF-?B1 and Attenuates Macrophage Activation *Prostaglandin E Receptor Type 4-associated Protein Interacts Directly with NF-?B1 and Attenuates Macrophage Activation * S⃞

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M709663200

EPRAP overexpression attenuates NF-κB activation and nuclear translocation induced by proinflammatory stimuli. A , EPRAP overexpression attenuates NF-κB activation induced by various stimuli in a dose-dependent manner. NF-κB reporter gene analyses were performed using HEK293 cells cotransfected with the secreted alkaline phosphatase reporter plasmid, LacZ expression plasmid, and increasing amounts of human EPRAP expression construct ( hEPRAP ) as described under “Experimental Procedures.” Transfection efficiency was normalized by β-galactosidase (β- Gal ) activity. Data are representative of three independent experiments. Results are expressed relative to the values of unstimulated cells in mean ± S.E. #, p
Figure Legend Snippet: EPRAP overexpression attenuates NF-κB activation and nuclear translocation induced by proinflammatory stimuli. A , EPRAP overexpression attenuates NF-κB activation induced by various stimuli in a dose-dependent manner. NF-κB reporter gene analyses were performed using HEK293 cells cotransfected with the secreted alkaline phosphatase reporter plasmid, LacZ expression plasmid, and increasing amounts of human EPRAP expression construct ( hEPRAP ) as described under “Experimental Procedures.” Transfection efficiency was normalized by β-galactosidase (β- Gal ) activity. Data are representative of three independent experiments. Results are expressed relative to the values of unstimulated cells in mean ± S.E. #, p

Techniques Used: Over Expression, Activation Assay, Translocation Assay, Plasmid Preparation, Expressing, Construct, Transfection, Activity Assay

17) Product Images from "The proapoptotic function of Drosophila Hid is conserved in mammalian cells"

Article Title: The proapoptotic function of Drosophila Hid is conserved in mammalian cells

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

Hid localizes to the mitochondria. HeLa cells were transfected with pcDNA3 Hid. Twenty-four hours posttransfection, the cells were stained with 100 nM MitoTracker for 30 minutes, fixed, and immunostained with a mAb to Hid (CL1C3) and a FITC-conjugated secondary antibody. A – C shows a single HeLa cell visualized with indirect fluorescence microscopy filtered to allow FITC ( A ), MitoTracker ( B ), or both ( C ) to be seen. ( D ) Enlarged detail of the area indicated by the white box in C .
Figure Legend Snippet: Hid localizes to the mitochondria. HeLa cells were transfected with pcDNA3 Hid. Twenty-four hours posttransfection, the cells were stained with 100 nM MitoTracker for 30 minutes, fixed, and immunostained with a mAb to Hid (CL1C3) and a FITC-conjugated secondary antibody. A – C shows a single HeLa cell visualized with indirect fluorescence microscopy filtered to allow FITC ( A ), MitoTracker ( B ), or both ( C ) to be seen. ( D ) Enlarged detail of the area indicated by the white box in C .

Techniques Used: Transfection, Staining, Fluorescence, Microscopy

Hid induces apoptotic morphology in HeLa cells. HeLa cells were transiently transfected with 0.5 μg of pcDNA3 empty vector ( A ) or 0.5 μg of pcDNA3 Hid ( B ) along with 0.5 μg of pCMV-lacZ. Twenty-four hours posttransfection, the cells were fixed, stained with X-Gal, and observed by using light microscopy. ( C – E ) HeLa cells were transfected with 0.5 μg of pcDNA3 Hid along with 0.5 μg of pEGFP-CMV. At 24 hours, they were fixed, and the cells were stained with Hoechst dye no. 33358 (5 μg/ml) for 30 minutes, washed, and observed by using fluorescence microscopy on a FITC filter set ( C ) and a blue filterset ( D ). ( E ) Artificially merged images. ( F ) Quantification of apoptosis: The indicated cell types were transfected with 0.5 μg of pcDNA3 Hid or pcDNA3 along with a 0.5 μg of pCMV-lacZ. Twenty-four hours posttransfection, the cells were fixed and stained with X-Gal. Two hundred blue cells were counted in each of two duplicate transfections in three independent experiments. The fraction of blue stained cells with apoptotic morphology as a percentage of all blue-stained cells was calculated. The percentage is expressed as the mean ± SEM.
Figure Legend Snippet: Hid induces apoptotic morphology in HeLa cells. HeLa cells were transiently transfected with 0.5 μg of pcDNA3 empty vector ( A ) or 0.5 μg of pcDNA3 Hid ( B ) along with 0.5 μg of pCMV-lacZ. Twenty-four hours posttransfection, the cells were fixed, stained with X-Gal, and observed by using light microscopy. ( C – E ) HeLa cells were transfected with 0.5 μg of pcDNA3 Hid along with 0.5 μg of pEGFP-CMV. At 24 hours, they were fixed, and the cells were stained with Hoechst dye no. 33358 (5 μg/ml) for 30 minutes, washed, and observed by using fluorescence microscopy on a FITC filter set ( C ) and a blue filterset ( D ). ( E ) Artificially merged images. ( F ) Quantification of apoptosis: The indicated cell types were transfected with 0.5 μg of pcDNA3 Hid or pcDNA3 along with a 0.5 μg of pCMV-lacZ. Twenty-four hours posttransfection, the cells were fixed and stained with X-Gal. Two hundred blue cells were counted in each of two duplicate transfections in three independent experiments. The fraction of blue stained cells with apoptotic morphology as a percentage of all blue-stained cells was calculated. The percentage is expressed as the mean ± SEM.

Techniques Used: Transfection, Plasmid Preparation, Staining, Light Microscopy, Fluorescence, Microscopy

18) Product Images from "Functional Characterization of Zebrafish (Danio rerio) Bcl10"

Article Title: Functional Characterization of Zebrafish (Danio rerio) Bcl10

Journal: PLoS ONE

doi: 10.1371/journal.pone.0122365

zBcl10 activates NF-κB in zebrafish cells. A) PAC2 cells were transiently cotransfected with expression vectors encoding for the indicated polypeptides, together with pNF-κB-luc and pRSV-βgal reporter vectors. The total amount of transfected plasmidic DNA was maintained constant by adding empty vector. 24 hrs after transfection, cell lysates were prepared and luciferase activity was measured. Data shown (mean + SEM, n = 9) represent relative luciferase activity normalized on β-galactosidase activity and is representative of six independent experiments done in triplicate. Statistical analysis was performed by Student's t test; a p value of
Figure Legend Snippet: zBcl10 activates NF-κB in zebrafish cells. A) PAC2 cells were transiently cotransfected with expression vectors encoding for the indicated polypeptides, together with pNF-κB-luc and pRSV-βgal reporter vectors. The total amount of transfected plasmidic DNA was maintained constant by adding empty vector. 24 hrs after transfection, cell lysates were prepared and luciferase activity was measured. Data shown (mean + SEM, n = 9) represent relative luciferase activity normalized on β-galactosidase activity and is representative of six independent experiments done in triplicate. Statistical analysis was performed by Student's t test; a p value of

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

zBcl10 activates NF-κB. A) HEK293 cells were transiently cotransfected with an expression vector empy ( vector ) or encoding for the indicated polypeptides, together with pNF-κB-luc and pRSV-βgal reporter vectors. The total amount of transfected plasmidic DNA was maintained constant by adding empty vector. 16 hrs after transfection, cell lysates were prepared and luciferase activity was measured. In the bottom panels, a fraction of the cell lysates were analyzed by immunoblot to monitor protein expression. Data shown (mean + SEM, n = 9) represent relative luciferase activity normalized on β-galactosidase activity and is representative of six independent experiments done in triplicate. Statistical analysis was performed by Student's t test; a p value of
Figure Legend Snippet: zBcl10 activates NF-κB. A) HEK293 cells were transiently cotransfected with an expression vector empy ( vector ) or encoding for the indicated polypeptides, together with pNF-κB-luc and pRSV-βgal reporter vectors. The total amount of transfected plasmidic DNA was maintained constant by adding empty vector. 16 hrs after transfection, cell lysates were prepared and luciferase activity was measured. In the bottom panels, a fraction of the cell lysates were analyzed by immunoblot to monitor protein expression. Data shown (mean + SEM, n = 9) represent relative luciferase activity normalized on β-galactosidase activity and is representative of six independent experiments done in triplicate. Statistical analysis was performed by Student's t test; a p value of

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

19) Product Images from "Multifunctional Role of the Pitx2 Homeodomain Protein C-Terminal Tail"

Article Title: Multifunctional Role of the Pitx2 Homeodomain Protein C-Terminal Tail

Journal: Molecular and Cellular Biology

doi:

Transcriptional activation of a TK- bicoid -luciferase reporter by Pitx2 and Pitx2 truncations in COS-7 cells. (A) COS-7 cells were transfected with either the TK- bicoid -luciferase reporter gene containing four copies of the Pitx2 binding site (dashed boxes) or the parental TK-luciferase reporter without the bicoid sites. The cells were cotransfected with either the CMV Pitx2, Pitx2CΔ39, Pitx2CΔ173, Pitx2NΔ16, or Pitx2NΔ38 expression plasmid or the CMV plasmid without Pitx2 (−). To control for transfection efficiency, all transfections included the CMV β-galactosidase reporter. Cells were incubated for 24 h and then were assayed for luciferase and β-galactosidase activities. The activities are shown as mean fold activations compared to that of TK- bicoid -luciferase without Pitx2 expression (± standard errors of the means from four independent experiments). The mean TK- bicoid -luciferase activity with Pitx2 expression was about 15,000 light units per 15 μg of protein, and the β-galactosidase activity was about 100,000 light units per 15 μg of protein. (B) Proteins were expressed in mammalian cells with a C-terminal myc epitope and detected by using a c-myc monoclonal antibody (9E10; Santa Cruz). Specific protein bands are denoted with an asterisk. (C) Quantitation of 6 to 10 independent Western blot experiments. Error bars are standard errors of the mean. Specific band intensities from the Western blots were measured by using NIH Image, with the bundled macros provided for gel analysis to measure band densities relative to the average background. Measurements are reported as relative intensity units.
Figure Legend Snippet: Transcriptional activation of a TK- bicoid -luciferase reporter by Pitx2 and Pitx2 truncations in COS-7 cells. (A) COS-7 cells were transfected with either the TK- bicoid -luciferase reporter gene containing four copies of the Pitx2 binding site (dashed boxes) or the parental TK-luciferase reporter without the bicoid sites. The cells were cotransfected with either the CMV Pitx2, Pitx2CΔ39, Pitx2CΔ173, Pitx2NΔ16, or Pitx2NΔ38 expression plasmid or the CMV plasmid without Pitx2 (−). To control for transfection efficiency, all transfections included the CMV β-galactosidase reporter. Cells were incubated for 24 h and then were assayed for luciferase and β-galactosidase activities. The activities are shown as mean fold activations compared to that of TK- bicoid -luciferase without Pitx2 expression (± standard errors of the means from four independent experiments). The mean TK- bicoid -luciferase activity with Pitx2 expression was about 15,000 light units per 15 μg of protein, and the β-galactosidase activity was about 100,000 light units per 15 μg of protein. (B) Proteins were expressed in mammalian cells with a C-terminal myc epitope and detected by using a c-myc monoclonal antibody (9E10; Santa Cruz). Specific protein bands are denoted with an asterisk. (C) Quantitation of 6 to 10 independent Western blot experiments. Error bars are standard errors of the mean. Specific band intensities from the Western blots were measured by using NIH Image, with the bundled macros provided for gel analysis to measure band densities relative to the average background. Measurements are reported as relative intensity units.

Techniques Used: Activation Assay, Luciferase, Transfection, Binding Assay, Expressing, Plasmid Preparation, Incubation, Activity Assay, Quantitation Assay, Western Blot

Pitx2 C-terminal truncations reduce the Pit-1 synergistic transactivation of the prolactin promoter. COS-7 cells were transfected with the prolactin 2.5-luciferase reporter gene and cotransfected with either the CMV Pitx2, Pitx2CΔ39, Pitx2NΔ16, Pitx2NΔ38, and Pitx2HD expression plasmids (+) or the CMV plasmid without Pitx2 (−). Pit-1 was cotransfected with the expression plasmids. To control for transfection efficiency, all transfections included the CMV β-galactosidase reporter. Cells were incubated for 24 h and then assayed for luciferase and β-galactosidase activities. The activities are shown as mean fold activations compared to activation of prolactin 2.5-luciferase without Pitx2 expression (± standard errors of the means from three independent experiments).
Figure Legend Snippet: Pitx2 C-terminal truncations reduce the Pit-1 synergistic transactivation of the prolactin promoter. COS-7 cells were transfected with the prolactin 2.5-luciferase reporter gene and cotransfected with either the CMV Pitx2, Pitx2CΔ39, Pitx2NΔ16, Pitx2NΔ38, and Pitx2HD expression plasmids (+) or the CMV plasmid without Pitx2 (−). Pit-1 was cotransfected with the expression plasmids. To control for transfection efficiency, all transfections included the CMV β-galactosidase reporter. Cells were incubated for 24 h and then assayed for luciferase and β-galactosidase activities. The activities are shown as mean fold activations compared to activation of prolactin 2.5-luciferase without Pitx2 expression (± standard errors of the means from three independent experiments).

Techniques Used: Transfection, Luciferase, Expressing, Plasmid Preparation, Incubation, Activation Assay

20) Product Images from "Advanced glycation end products induce cell cycle arrest and proinflammatory changes in osteoarthritic fibroblast-like synovial cells"

Article Title: Advanced glycation end products induce cell cycle arrest and proinflammatory changes in osteoarthritic fibroblast-like synovial cells

Journal: Arthritis Research & Therapy

doi: 10.1186/ar2807

In vivo transactivation of NFκB. A luciferase reporter plasmid containing consensus binding sites for nuclear factor kappa B (NFκB) was transfected in two different fibroblast-like synovial cell (FLS) lines. Advanced glycation end products-modified (AGE)-BSA significantly enhanced NFκB transactivation compared with control-BSA (Co-BSA; * P
Figure Legend Snippet: In vivo transactivation of NFκB. A luciferase reporter plasmid containing consensus binding sites for nuclear factor kappa B (NFκB) was transfected in two different fibroblast-like synovial cell (FLS) lines. Advanced glycation end products-modified (AGE)-BSA significantly enhanced NFκB transactivation compared with control-BSA (Co-BSA; * P

Techniques Used: In Vivo, Luciferase, Plasmid Preparation, Binding Assay, Transfection, Modification

21) Product Images from "Down-Regulation of Cyclin D1 Expression by Prostaglandin A2 Is Mediated by Enhanced Cyclin D1 mRNA Turnover"

Article Title: Down-Regulation of Cyclin D1 Expression by Prostaglandin A2 Is Mediated by Enhanced Cyclin D1 mRNA Turnover

Journal: Molecular and Cellular Biology

doi:

Influence of the K12 region of the cyclin D1 mRNA on the expression of a chimeric EGFP reporter construct after PGA 2 treatment. Five micrograms of each pTRE-d2EGFP, pTRE-d2EGFP-CR, or pTRE-d2EGFP-K12 plasmid was transiently transfected into H2 cells (previously transfected with pTet and selected based on its strong induced and high doxycycline-dependent repression of reporter gene expression), along with 1 μg of β-galactosidase reporter as a control. Twenty hours after transfection, cells were treated either with doxycycline alone (control) or with a combination of doxycycline and PGA 2 (30 μM, PGA 2 ). At the times indicated following addition of the drug(s), RNA was prepared for Northern blot analysis; assessment of the expression levels of the EGFP, EGFP-CR, or EGFP-K12 transcripts was followed by that of cyclin D1 expression (not shown) and 18S rRNA, through sequential rounds of stripping and hybridization. Representative Northerns are shown. Graphs depict the relative abundance of each EGFP-derived transcript at various times after addition of doxycycline (time 0, 100%), represented on a logarithmic scale. Dashed line, 50% mRNA, which served to obtain the half-life values for each transcript.
Figure Legend Snippet: Influence of the K12 region of the cyclin D1 mRNA on the expression of a chimeric EGFP reporter construct after PGA 2 treatment. Five micrograms of each pTRE-d2EGFP, pTRE-d2EGFP-CR, or pTRE-d2EGFP-K12 plasmid was transiently transfected into H2 cells (previously transfected with pTet and selected based on its strong induced and high doxycycline-dependent repression of reporter gene expression), along with 1 μg of β-galactosidase reporter as a control. Twenty hours after transfection, cells were treated either with doxycycline alone (control) or with a combination of doxycycline and PGA 2 (30 μM, PGA 2 ). At the times indicated following addition of the drug(s), RNA was prepared for Northern blot analysis; assessment of the expression levels of the EGFP, EGFP-CR, or EGFP-K12 transcripts was followed by that of cyclin D1 expression (not shown) and 18S rRNA, through sequential rounds of stripping and hybridization. Representative Northerns are shown. Graphs depict the relative abundance of each EGFP-derived transcript at various times after addition of doxycycline (time 0, 100%), represented on a logarithmic scale. Dashed line, 50% mRNA, which served to obtain the half-life values for each transcript.

Techniques Used: Expressing, Construct, Plasmid Preparation, Transfection, Northern Blot, Stripping Membranes, Hybridization, Derivative Assay

22) Product Images from "Green Tea (−)-Epigallotocatechin-3-Gallate Induces PGC-1α Gene Expression in HepG2 Cells and 3T3-L1 Adipocytes"

Article Title: Green Tea (−)-Epigallotocatechin-3-Gallate Induces PGC-1α Gene Expression in HepG2 Cells and 3T3-L1 Adipocytes

Journal: Preventive Nutrition and Food Science

doi: 10.3746/pnf.2016.21.1.62

Effects of EGCG on the promoter activity of PGC-1α in HepG2 cells (A) and 3T3-L1 adipocytes (B). HepG2 cells and differentiated 3T3-L1 adipocytes were transfected with PGC-1α (−970/+412 bp)/luciferase reporter gene and pCMV-β galactosidase, and were then incubated in serum-free media with the indicated concentrations of EGCG, from 0 to 10 μmol/L, for 40 h. Luciferase activity was calculated in relative light units and normalized to the β-galactosidase activity. Values are expressed as mean±SEM (n=3) of three independent experiments. * P
Figure Legend Snippet: Effects of EGCG on the promoter activity of PGC-1α in HepG2 cells (A) and 3T3-L1 adipocytes (B). HepG2 cells and differentiated 3T3-L1 adipocytes were transfected with PGC-1α (−970/+412 bp)/luciferase reporter gene and pCMV-β galactosidase, and were then incubated in serum-free media with the indicated concentrations of EGCG, from 0 to 10 μmol/L, for 40 h. Luciferase activity was calculated in relative light units and normalized to the β-galactosidase activity. Values are expressed as mean±SEM (n=3) of three independent experiments. * P

Techniques Used: Activity Assay, Pyrolysis Gas Chromatography, Transfection, Luciferase, Incubation

23) Product Images from "Both Linear and Discontinuous Ribosome Scanning Are Used for Translation Initiation from Bicistronic Human Immunodeficiency Virus Type 1 env mRNAs ▿ mRNAs ▿ †"

Article Title: Both Linear and Discontinuous Ribosome Scanning Are Used for Translation Initiation from Bicistronic Human Immunodeficiency Virus Type 1 env mRNAs ▿ mRNAs ▿ †

Journal: Journal of Virology

doi: 10.1128/JVI.01028-06

Increasing the strength of or removing the Rev AUG modulates Vpu expression. (A) Mutations in the env2 cDNA constructs that either strengthened the Rev AUG codon (underlined; RevK) or removed it (Rev − ). (B and C) HeLa cells were transfected with the env cDNA constructs, pCMVTat2X, pEGFP, and pDM128 without (B) or with (C) pLTR rev1 . Rev activity was measured by CAT assay. Env and Vpu were detected by immunoblotting followed by densitometry analysis, and the percentage of expression relative to the expression of the env1 R38 mutant (E1R38; set at 100%) is graphed. wt, wild type; Mk, mock (no env or pLTR rev1 ); E1 and E2, pDR env1 and pDR env2 ; E2K, env2 cDNA construct expressing RevK mutation; E2R38, env2 cDNA construct expressing R38 mutation; E2KR38, env2 cDNA construct expressing RevK and R38 mutations; E2R−, env2 cDNA construct expressing Rev − mutation.
Figure Legend Snippet: Increasing the strength of or removing the Rev AUG modulates Vpu expression. (A) Mutations in the env2 cDNA constructs that either strengthened the Rev AUG codon (underlined; RevK) or removed it (Rev − ). (B and C) HeLa cells were transfected with the env cDNA constructs, pCMVTat2X, pEGFP, and pDM128 without (B) or with (C) pLTR rev1 . Rev activity was measured by CAT assay. Env and Vpu were detected by immunoblotting followed by densitometry analysis, and the percentage of expression relative to the expression of the env1 R38 mutant (E1R38; set at 100%) is graphed. wt, wild type; Mk, mock (no env or pLTR rev1 ); E1 and E2, pDR env1 and pDR env2 ; E2K, env2 cDNA construct expressing RevK mutation; E2R38, env2 cDNA construct expressing R38 mutation; E2KR38, env2 cDNA construct expressing RevK and R38 mutations; E2R−, env2 cDNA construct expressing Rev − mutation.

Techniques Used: Expressing, Construct, Transfection, Activity Assay, Mutagenesis

Similar levels of Env but different levels of Vpu coexpressed by the env isoforms with saturating Rev in trans . (A) Rev activity 40 h after triplicate transfections of HeLa cells with pLTR rev1 (8 μg) along with the env cDNA construct, pCMVTat2x, pEGFP, and pDM128 was determined by CAT assays. (B and C) Samples normalized for transfection efficiency by using GFP were immunoblotted for gp160 Env (B) and Vpu (C), with representative blots shown. (D) The density of the Vpu band was divided by the density of the Env band for each env isoform, and the average ratio from three independent experiments was graphed. Mk, mock (no env or pLTR rev1 ) transfection control; Rev, pLTR rev1 (no env ) transfection control; E1 to E14, plasmids expressing isoforms env1 to env14 .
Figure Legend Snippet: Similar levels of Env but different levels of Vpu coexpressed by the env isoforms with saturating Rev in trans . (A) Rev activity 40 h after triplicate transfections of HeLa cells with pLTR rev1 (8 μg) along with the env cDNA construct, pCMVTat2x, pEGFP, and pDM128 was determined by CAT assays. (B and C) Samples normalized for transfection efficiency by using GFP were immunoblotted for gp160 Env (B) and Vpu (C), with representative blots shown. (D) The density of the Vpu band was divided by the density of the Env band for each env isoform, and the average ratio from three independent experiments was graphed. Mk, mock (no env or pLTR rev1 ) transfection control; Rev, pLTR rev1 (no env ) transfection control; E1 to E14, plasmids expressing isoforms env1 to env14 .

Techniques Used: Activity Assay, Transfection, Construct, Expressing

Adding an upstream, strong Kozak AUG codon in exon 2 of the env isoforms does not diminish Env expression. (A) Mutations in exon 2 of the env cDNA constructs that introduced either a strong Kozak AUG codon (KAUG) or the same mutations minus the AUG codon (KAGG). A UAA translation termination motif was included at codon 4 to avoid aberrant protein synthesis. (B) HeLa cells transfected with the env cDNA constructs, pCMVTat2X, pLTR rev1 , and pEGFP were analyzed for Env and Vpu by immunoblotting and densitometry. The percentage of expression relative to that of the env5 construct (E5; set at 100%) is graphed. wt, wild type; E6, pDR env6 ; E5KAGG and E6KAGG, env5 and env6 constructs without the Kozak AUG codon; E5KAUG and E6KAUG, env5 and env6 constructs with the Kozak AUG codon.
Figure Legend Snippet: Adding an upstream, strong Kozak AUG codon in exon 2 of the env isoforms does not diminish Env expression. (A) Mutations in exon 2 of the env cDNA constructs that introduced either a strong Kozak AUG codon (KAUG) or the same mutations minus the AUG codon (KAGG). A UAA translation termination motif was included at codon 4 to avoid aberrant protein synthesis. (B) HeLa cells transfected with the env cDNA constructs, pCMVTat2X, pLTR rev1 , and pEGFP were analyzed for Env and Vpu by immunoblotting and densitometry. The percentage of expression relative to that of the env5 construct (E5; set at 100%) is graphed. wt, wild type; E6, pDR env6 ; E5KAGG and E6KAGG, env5 and env6 constructs without the Kozak AUG codon; E5KAUG and E6KAUG, env5 and env6 constructs with the Kozak AUG codon.

Techniques Used: Expressing, Construct, Transfection

Rev R38 mutant env isoforms express different levels of Vpu but similar levels of Env irrespective of the Rev titer. (A) The R38 mutation introduces a premature translation termination codon corresponding to amino acid 38 in the second Rev exon of the env cDNA constructs. (B) The activity of Rev R38 env isoform mutants was measured by CAT assays 40 h after the cotransfection of HeLa cells with pCMVTat2X, pEGFP, and pDM128 with or without pLTR rev1 . (C) Densitometry analysis of Env and Vpu expression from the env cDNA constructs. (D) Immunoblot assay of gp160 Env and Vpu synthesized in HeLa cells cotransfected with env cDNA, pCMVTat2X, pEGFP, pDM128, and increasing amounts of pLTR rev1 . wt, wild-type sequence; R, R38 mutant sequence; Mk, mock (no env or pLTR rev1 ) transfection control; NL, HIV NL4.3 proviral plasmid control; E1R to E4R, env1 to env4 R38 mutant plasmids.
Figure Legend Snippet: Rev R38 mutant env isoforms express different levels of Vpu but similar levels of Env irrespective of the Rev titer. (A) The R38 mutation introduces a premature translation termination codon corresponding to amino acid 38 in the second Rev exon of the env cDNA constructs. (B) The activity of Rev R38 env isoform mutants was measured by CAT assays 40 h after the cotransfection of HeLa cells with pCMVTat2X, pEGFP, and pDM128 with or without pLTR rev1 . (C) Densitometry analysis of Env and Vpu expression from the env cDNA constructs. (D) Immunoblot assay of gp160 Env and Vpu synthesized in HeLa cells cotransfected with env cDNA, pCMVTat2X, pEGFP, pDM128, and increasing amounts of pLTR rev1 . wt, wild-type sequence; R, R38 mutant sequence; Mk, mock (no env or pLTR rev1 ) transfection control; NL, HIV NL4.3 proviral plasmid control; E1R to E4R, env1 to env4 R38 mutant plasmids.

Techniques Used: Mutagenesis, Construct, Activity Assay, Cotransfection, Expressing, Synthesized, Sequencing, Transfection, Plasmid Preparation

Removing upstream AUG codons and changing Vpu ORF length does not alter consistent Env expression. (A) Mutations in the env cDNA constructs that removed the upstream Rev and/or Vpu AUG codon (underlined; Rev− and/or Vpu−) or shortened the Vpu ORF at the position corresponding to amino acid 2 (Vpu2) or 35 (ΔU35). Numbers indicate amino acid positions corresponding to the codons. (B and C) HeLa cells were transfected with the env cDNA constructs, pCMVTat2X, pLTR rev1 , and pEGFP. Env and Vpu expression was analyzed by immunoblotting and then densitometry. The percentage of expression relative to that of the env1 R38 mutant (E1R; set at 100%) is graphed for the env isoforms lacking upstream AUG codons (B) or containing altered Vpu ORF lengths (C). wt, wild type; Mk, mock (no env ); E1R to E3R, pDR env1 to pDR env3 cDNA constructs containing the Rev R38 premature termination codon mutation; E2−, env2 cDNA construct expressing the Rev − mutation; E2V−, env2 cDNA construct expressing the Vpu − mutation; E2R−V−, env2 cDNA construct expressing the Rev − and Vpu − mutations; E1RΔU35 to E3RΔU35, env1 to env3 R38 mutant cDNA constructs with the Vpu ORF shortened at the position corresponding to amino acid 35; E1RVpu2 to E3RVpu2, env1 to env3 R38 mutant cDNA constructs with the Vpu ORF shortened at the position corresponding to amino acid 2.
Figure Legend Snippet: Removing upstream AUG codons and changing Vpu ORF length does not alter consistent Env expression. (A) Mutations in the env cDNA constructs that removed the upstream Rev and/or Vpu AUG codon (underlined; Rev− and/or Vpu−) or shortened the Vpu ORF at the position corresponding to amino acid 2 (Vpu2) or 35 (ΔU35). Numbers indicate amino acid positions corresponding to the codons. (B and C) HeLa cells were transfected with the env cDNA constructs, pCMVTat2X, pLTR rev1 , and pEGFP. Env and Vpu expression was analyzed by immunoblotting and then densitometry. The percentage of expression relative to that of the env1 R38 mutant (E1R; set at 100%) is graphed for the env isoforms lacking upstream AUG codons (B) or containing altered Vpu ORF lengths (C). wt, wild type; Mk, mock (no env ); E1R to E3R, pDR env1 to pDR env3 cDNA constructs containing the Rev R38 premature termination codon mutation; E2−, env2 cDNA construct expressing the Rev − mutation; E2V−, env2 cDNA construct expressing the Vpu − mutation; E2R−V−, env2 cDNA construct expressing the Rev − and Vpu − mutations; E1RΔU35 to E3RΔU35, env1 to env3 R38 mutant cDNA constructs with the Vpu ORF shortened at the position corresponding to amino acid 35; E1RVpu2 to E3RVpu2, env1 to env3 R38 mutant cDNA constructs with the Vpu ORF shortened at the position corresponding to amino acid 2.

Techniques Used: Expressing, Construct, Transfection, Mutagenesis

24) Product Images from "Treatment with Caffeic Acid and Resveratrol Alleviates Oxidative Stress Induced Neurotoxicity in Cell and Drosophila Models of Spinocerebellar Ataxia Type3"

Article Title: Treatment with Caffeic Acid and Resveratrol Alleviates Oxidative Stress Induced Neurotoxicity in Cell and Drosophila Models of Spinocerebellar Ataxia Type3

Journal: Scientific Reports

doi: 10.1038/s41598-017-11839-0

Effects of CA and Res in tBH-treated SK-N-SH-MJD78 cells transfected with dominant-negative mutant IκB-α. Cells were transiently transfected with IκB-α wild-type (WT) or DNM IκB-α as well as with or without reporter genes of p53-Luc or pNF-κB-Luc for 16 h and were then treated with either vehicle control or tBH plus CA or Res. (A) Phosphorylated and total p53 and IκB-α as well as nuclear p53 and p65 (3-h and 4-h treatments for p53 and NF-κB activation, respectively). (B) Protein expression of mutant and normal ataxin-3 and Hsp27. (C) Protein expression of Bax, Bcl-2, and pro and cleaved caspase 3 and PARP. (D) Levels of NF-κB and p53 reporter gene activities, protein aggregates, cell apoptosis rates, and caspase 3 activity. Data are the mean ± SD. Within treatments with the same plasmid transfection, data are expressed as the percentage of the SK-N-SH-MJD78 cells treated with tBH alone, and values not having the same letter are significantly different (p
Figure Legend Snippet: Effects of CA and Res in tBH-treated SK-N-SH-MJD78 cells transfected with dominant-negative mutant IκB-α. Cells were transiently transfected with IκB-α wild-type (WT) or DNM IκB-α as well as with or without reporter genes of p53-Luc or pNF-κB-Luc for 16 h and were then treated with either vehicle control or tBH plus CA or Res. (A) Phosphorylated and total p53 and IκB-α as well as nuclear p53 and p65 (3-h and 4-h treatments for p53 and NF-κB activation, respectively). (B) Protein expression of mutant and normal ataxin-3 and Hsp27. (C) Protein expression of Bax, Bcl-2, and pro and cleaved caspase 3 and PARP. (D) Levels of NF-κB and p53 reporter gene activities, protein aggregates, cell apoptosis rates, and caspase 3 activity. Data are the mean ± SD. Within treatments with the same plasmid transfection, data are expressed as the percentage of the SK-N-SH-MJD78 cells treated with tBH alone, and values not having the same letter are significantly different (p

Techniques Used: Transfection, Dominant Negative Mutation, Activation Assay, Expressing, Mutagenesis, Activity Assay, Plasmid Preparation

25) Product Images from "Requirements of Fission Yeast Septins for Complex Formation, Localization, and Function D⃞"

Article Title: Requirements of Fission Yeast Septins for Complex Formation, Localization, and Function D⃞

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E04-07-0640

Protein-protein interactions mapped between septin complex components. (A and H) Models of S. pombe septin interactions. (B) The PJ69-4A strain was transformed with pGBT9 or pGBT9 spn3 , and pGAD424 or pGAD424 carrying spn1, spn2 , or spn4 , which are shown schematically. (C) β-galactosidase activity (represented in relative light units) of the stains described in B. (D) The PJ69-4A stain was transformed with pGBT9 or pGBT9 spn1 and pGAD424 or pGAD424 carrying spn2, spn3, or spn4 , which are shown schematically. (E) β-galactosidase activity (represented in relative light units) of the stains described in D. (F) The PJ69-4A stain was transformed with pGBT9 or pGBT9 spn2 and pGAD424 or pGAD424 carrying spn4 , which are shown schematically. (G) β-galactosidase activity (represented in relative light units) of the stains described in D. Asterisk indicates positive interactions.
Figure Legend Snippet: Protein-protein interactions mapped between septin complex components. (A and H) Models of S. pombe septin interactions. (B) The PJ69-4A strain was transformed with pGBT9 or pGBT9 spn3 , and pGAD424 or pGAD424 carrying spn1, spn2 , or spn4 , which are shown schematically. (C) β-galactosidase activity (represented in relative light units) of the stains described in B. (D) The PJ69-4A stain was transformed with pGBT9 or pGBT9 spn1 and pGAD424 or pGAD424 carrying spn2, spn3, or spn4 , which are shown schematically. (E) β-galactosidase activity (represented in relative light units) of the stains described in D. (F) The PJ69-4A stain was transformed with pGBT9 or pGBT9 spn2 and pGAD424 or pGAD424 carrying spn4 , which are shown schematically. (G) β-galactosidase activity (represented in relative light units) of the stains described in D. Asterisk indicates positive interactions.

Techniques Used: Transformation Assay, Activity Assay, Staining

26) Product Images from "A recurrent WARS mutation is a novel cause of autosomal dominant distal hereditary motor neuropathy"

Article Title: A recurrent WARS mutation is a novel cause of autosomal dominant distal hereditary motor neuropathy

Journal: Brain

doi: 10.1093/brain/awx058

In vitro characterization of wild-type and His257Arg (H257R) mutant TrpRS proteins. ( A ) β-Gal reporter assay and ( B ) firefly luciferase reporter assay demonstrating that H257R TrpRS has a dominant-negative effect on protein synthesis. HEK293 cells co-transfected with β-Gal or firefly luciferase reporter plasmids, along with different ratios of wild-type and H257R TrpRS expression plasmids were lysed and assayed for β-Gal or firefly luciferase activities at 48 h after transfection. The error bars indicate standard errors of the mean ( n = 3) and asterisks indicate statistically significant differences (** P
Figure Legend Snippet: In vitro characterization of wild-type and His257Arg (H257R) mutant TrpRS proteins. ( A ) β-Gal reporter assay and ( B ) firefly luciferase reporter assay demonstrating that H257R TrpRS has a dominant-negative effect on protein synthesis. HEK293 cells co-transfected with β-Gal or firefly luciferase reporter plasmids, along with different ratios of wild-type and H257R TrpRS expression plasmids were lysed and assayed for β-Gal or firefly luciferase activities at 48 h after transfection. The error bars indicate standard errors of the mean ( n = 3) and asterisks indicate statistically significant differences (** P

Techniques Used: In Vitro, Mutagenesis, Reporter Assay, Luciferase, Dominant Negative Mutation, Transfection, Expressing

27) Product Images from "Critical Role Played by Cyclin D3 in the MyoD-Mediated Arrest of Cell Cycle during Myoblast Differentiation"

Article Title: Critical Role Played by Cyclin D3 in the MyoD-Mediated Arrest of Cell Cycle during Myoblast Differentiation

Journal: Molecular and Cellular Biology

doi:

Effect of ectopic expression of cyclin D3 on MCK expression in growing and differentiating C2 myoblasts. Proliferating C2 myoblasts were transfected with 0.5 μg of the MCK luc reporter plasmid in the presence of the indicated amount of the cyclin D3 expression construct (Rc/CMV-cycD3); the Rc/CMV expression vehicle without an insert was included to normalize DNA in all transfections. Eighteen hours after transfection, the cells were trypsinized; one half of the cells were plated onto 90-mm-diameter dishes, the other half were plated onto 60-mm-diameter dishes, and refed with growth medium. Parallel transfections were directly transferred to differentiation medium. After 72 h the cells in 90-mm-diameter dishes were subconfluent (A), those in 60-mm-diameter dishes were confluent (B), and those in differentiation medium were fully differentiated (C); at that time cells were collected and assayed for luciferase and β-galactosidase activities. Equal amounts of proteins from each cell lysate were also assayed for cyclin D3 expression by Western blotting. The results shown are from one representative experiment. MCK-luc activity is expressed relative to the levels detected in the absence of cyclin D3. The experiments were done in duplicate and repeated three times. The mean values (expressed as fold induction relative to the baseline values) and the standard deviations (S.D.) of these experiments are shown at the bottom.
Figure Legend Snippet: Effect of ectopic expression of cyclin D3 on MCK expression in growing and differentiating C2 myoblasts. Proliferating C2 myoblasts were transfected with 0.5 μg of the MCK luc reporter plasmid in the presence of the indicated amount of the cyclin D3 expression construct (Rc/CMV-cycD3); the Rc/CMV expression vehicle without an insert was included to normalize DNA in all transfections. Eighteen hours after transfection, the cells were trypsinized; one half of the cells were plated onto 90-mm-diameter dishes, the other half were plated onto 60-mm-diameter dishes, and refed with growth medium. Parallel transfections were directly transferred to differentiation medium. After 72 h the cells in 90-mm-diameter dishes were subconfluent (A), those in 60-mm-diameter dishes were confluent (B), and those in differentiation medium were fully differentiated (C); at that time cells were collected and assayed for luciferase and β-galactosidase activities. Equal amounts of proteins from each cell lysate were also assayed for cyclin D3 expression by Western blotting. The results shown are from one representative experiment. MCK-luc activity is expressed relative to the levels detected in the absence of cyclin D3. The experiments were done in duplicate and repeated three times. The mean values (expressed as fold induction relative to the baseline values) and the standard deviations (S.D.) of these experiments are shown at the bottom.

Techniques Used: Expressing, Transfection, Plasmid Preparation, Construct, Luciferase, Western Blot, Activity Assay

28) Product Images from "Functional characterization of the interferon regulatory element in the enhancer 1 region of the hepatitis B virus genome"

Article Title: Functional characterization of the interferon regulatory element in the enhancer 1 region of the hepatitis B virus genome

Journal: Nucleic Acids Research

doi:

Functional analysis of the HBV enhancer 1/X gene promoter ISRE/ IRE in the context of a minimal promoter. The XpIRE(1)TATALUC, XpIRE(4)TATALUC, XpIREmut(4)TATALUC and ISG15-ISRE(3)TATALUC constructs contain the XpIRE (enhancer 1/X gene promoter IRE), XpIREmut (enhancer 1/X gene promoter mutant IRE) and ISG15-ISRE (ISG15 promoter ISRE) double-stranded oligonucleotides cloned into the minimal promoter construct pHBVTATALUC, respectively. The number of copies of the oligonucleotide in the constructs is shown in parentheses in the construct name. The ISG54LUC construct contains the hamster ISG54 promoter spanning –429 to +31 controlling the level of expression of the luciferase reporter gene (39). The p[(AAGTGA)4]5tkΔ(–39)lucter construct contains five copies of a synthetic IRE upstream of the herpes simplex virus minimal thymidine kinase promoter that regulates the level of expression of the luciferase reporter gene (40). Relative activities of the constructs in HepG2 cells in the absence of treatment or ectopically expressed transcription factors (pCMV), in the presence of ectopically expressed interferon-stimulated gene factor 3 (ISGF3), after IFNα treatment (IFNalpha), in the presence of ectopically expressed ISGF3 with IFNα treatment (ISGF3+IFNalpha) and after poly(I)·poly(C) treatment are indicated. ISGF3 was expressed by co-transfecting the expression vectors pCMVSTAT1α, pCMVSTAT2 and pCMVISGF3γ p48. The transcriptional activities are reported relative to the ISG15-ISRE(3)LUC plasmid in the absence of any expression vector or treatment and is designated as having a relative activity of 1.0. The relative activities of the ISG54LUC construct under the various conditions were divided by 10 so that all constructs could be shown using the same scale. The standard deviation of the mean is indicated by an error bar. The internal control used to correct for transfection efficiencies was pCMVβ.
Figure Legend Snippet: Functional analysis of the HBV enhancer 1/X gene promoter ISRE/ IRE in the context of a minimal promoter. The XpIRE(1)TATALUC, XpIRE(4)TATALUC, XpIREmut(4)TATALUC and ISG15-ISRE(3)TATALUC constructs contain the XpIRE (enhancer 1/X gene promoter IRE), XpIREmut (enhancer 1/X gene promoter mutant IRE) and ISG15-ISRE (ISG15 promoter ISRE) double-stranded oligonucleotides cloned into the minimal promoter construct pHBVTATALUC, respectively. The number of copies of the oligonucleotide in the constructs is shown in parentheses in the construct name. The ISG54LUC construct contains the hamster ISG54 promoter spanning –429 to +31 controlling the level of expression of the luciferase reporter gene (39). The p[(AAGTGA)4]5tkΔ(–39)lucter construct contains five copies of a synthetic IRE upstream of the herpes simplex virus minimal thymidine kinase promoter that regulates the level of expression of the luciferase reporter gene (40). Relative activities of the constructs in HepG2 cells in the absence of treatment or ectopically expressed transcription factors (pCMV), in the presence of ectopically expressed interferon-stimulated gene factor 3 (ISGF3), after IFNα treatment (IFNalpha), in the presence of ectopically expressed ISGF3 with IFNα treatment (ISGF3+IFNalpha) and after poly(I)·poly(C) treatment are indicated. ISGF3 was expressed by co-transfecting the expression vectors pCMVSTAT1α, pCMVSTAT2 and pCMVISGF3γ p48. The transcriptional activities are reported relative to the ISG15-ISRE(3)LUC plasmid in the absence of any expression vector or treatment and is designated as having a relative activity of 1.0. The relative activities of the ISG54LUC construct under the various conditions were divided by 10 so that all constructs could be shown using the same scale. The standard deviation of the mean is indicated by an error bar. The internal control used to correct for transfection efficiencies was pCMVβ.

Techniques Used: Functional Assay, Construct, Mutagenesis, Clone Assay, Expressing, Luciferase, Plasmid Preparation, Activity Assay, Standard Deviation, Transfection

29) Product Images from "Differential regulation of insulin-like growth factor-I receptor gene expression by wild type and mutant androgen receptor in prostate cancer cells"

Article Title: Differential regulation of insulin-like growth factor-I receptor gene expression by wild type and mutant androgen receptor in prostate cancer cells

Journal: Molecular and cellular endocrinology

doi: 10.1016/j.mce.2010.04.017

Regulation of IGF-IR promoter activity by unligated and ligand-activated AR. P69 (A) and M12 (B) cells were cotransfected with the p(−476/+640)LUC IGF-IR promoter-luciferase reporter, along with an AR vector (or pcDNA3) and a β-gal vector.
Figure Legend Snippet: Regulation of IGF-IR promoter activity by unligated and ligand-activated AR. P69 (A) and M12 (B) cells were cotransfected with the p(−476/+640)LUC IGF-IR promoter-luciferase reporter, along with an AR vector (or pcDNA3) and a β-gal vector.

Techniques Used: Activity Assay, Luciferase, Plasmid Preparation

30) Product Images from "Glucocorticoid Receptor Interacting Protein-1 Restores Glucocorticoid Responsiveness in Steroid-Resistant Airway Structural Cells"

Article Title: Glucocorticoid Receptor Interacting Protein-1 Restores Glucocorticoid Responsiveness in Steroid-Resistant Airway Structural Cells

Journal:

doi: 10.1165/rcmb.2009-0239RC

GRIP-1 overexpression rescues GR transcriptional activation from cytokine-/IRF-1–dependent inhibition. ( A ) ASM cells co-transfected with GRE-luciferase reporter and either 2 μg of full-length GRIP-1 or pcDNA3 were exposed to TNF-α
Figure Legend Snippet: GRIP-1 overexpression rescues GR transcriptional activation from cytokine-/IRF-1–dependent inhibition. ( A ) ASM cells co-transfected with GRE-luciferase reporter and either 2 μg of full-length GRIP-1 or pcDNA3 were exposed to TNF-α

Techniques Used: Over Expression, Activation Assay, Inhibition, Transfection, Luciferase

31) Product Images from "Phosphorylation Regulates Functions of ZEB1 Transcription Factor"

Article Title: Phosphorylation Regulates Functions of ZEB1 Transcription Factor

Journal: Journal of cellular physiology

doi: 10.1002/jcp.25338

PMA/Iono treatment of cells inhibits ZEB1-mediated repression COS-7 and CHO-K1 cells were co-transfected and treated with PMA/Iono for 30 minutes as indicated in M M. (A) In the absence of cotransfected ZEB1, PMA/Iono has no effect. Results are expressed as mean ± S.E.M. of the fold luciferase activation normalized to the vector pGL3-basic. (B-D) The effect of kinase activation on ZEB1-mediated repression is tested with (B) the ZEB1 promoter, (C) the p73intr promoter, or (D) the CD4 promoter. Results are expressed as mean ± S.E.M. (n=4-6) of promoter activation. DMSO or PMA+Iono treatment without transfected ZEB1 is set as 100%. *P
Figure Legend Snippet: PMA/Iono treatment of cells inhibits ZEB1-mediated repression COS-7 and CHO-K1 cells were co-transfected and treated with PMA/Iono for 30 minutes as indicated in M M. (A) In the absence of cotransfected ZEB1, PMA/Iono has no effect. Results are expressed as mean ± S.E.M. of the fold luciferase activation normalized to the vector pGL3-basic. (B-D) The effect of kinase activation on ZEB1-mediated repression is tested with (B) the ZEB1 promoter, (C) the p73intr promoter, or (D) the CD4 promoter. Results are expressed as mean ± S.E.M. (n=4-6) of promoter activation. DMSO or PMA+Iono treatment without transfected ZEB1 is set as 100%. *P

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

Phosphorylation within ZD2 inhibits binding to DNA and transcriptional activity (A) Cartoons showing the ZEB1 structure representing its zinc finger domains (ZD1 and ZD2), the central homeodomain (HD) and an acidic domain (EE). The ZEB1 subclones used in different experiments are also illustrated. (B) EMSAs were performed with RRL programmed with ZD2 mRNA. ZD2-RRLs were incubated with [ 32 P]α4-integrin (lane 1), and ZEB1 R17 antibody (lane 2), or an anti-actin antibody (lane 3), or were competed by 20X excess of unlabelled α4-integrin oligonucleotide (lane 4). ZD2-programmed RRL was treated with buffer alone (lanes 5, 7) or CIP at 30°C (lane 6) or 37°C (lane 8). (C) Luciferase reporter assay in CHO-K1 cells showing the CD4 promoter-luciferase activity co-transfected with the expression vectors for full-length ZEB1 or wtZD2 after PMA/Iono incubation for 10 min. to 30 min. The results indicate mean ± S.E.M. (n=2).
Figure Legend Snippet: Phosphorylation within ZD2 inhibits binding to DNA and transcriptional activity (A) Cartoons showing the ZEB1 structure representing its zinc finger domains (ZD1 and ZD2), the central homeodomain (HD) and an acidic domain (EE). The ZEB1 subclones used in different experiments are also illustrated. (B) EMSAs were performed with RRL programmed with ZD2 mRNA. ZD2-RRLs were incubated with [ 32 P]α4-integrin (lane 1), and ZEB1 R17 antibody (lane 2), or an anti-actin antibody (lane 3), or were competed by 20X excess of unlabelled α4-integrin oligonucleotide (lane 4). ZD2-programmed RRL was treated with buffer alone (lanes 5, 7) or CIP at 30°C (lane 6) or 37°C (lane 8). (C) Luciferase reporter assay in CHO-K1 cells showing the CD4 promoter-luciferase activity co-transfected with the expression vectors for full-length ZEB1 or wtZD2 after PMA/Iono incubation for 10 min. to 30 min. The results indicate mean ± S.E.M. (n=2).

Techniques Used: Binding Assay, Activity Assay, Incubation, Luciferase, Reporter Assay, Transfection, Expressing

Phosphorylation of ZD2 alters nuclear localization (A) Confocal microscopy of CHO-K1 cells transfected with the pEGFP-GKLF, GFP-ZD2 or GFP-ZD2-NLS expression vectors treated as indicated. (B) .
Figure Legend Snippet: Phosphorylation of ZD2 alters nuclear localization (A) Confocal microscopy of CHO-K1 cells transfected with the pEGFP-GKLF, GFP-ZD2 or GFP-ZD2-NLS expression vectors treated as indicated. (B) .

Techniques Used: Confocal Microscopy, Transfection, Expressing

32) Product Images from "Polymorphism of the human ?1 immunoglobulin gene 3? enhancer hs1,2 and its relation to gene expression"

Article Title: Polymorphism of the human ?1 immunoglobulin gene 3? enhancer hs1,2 and its relation to gene expression

Journal: Immunology

doi: 10.1046/j.1365-2567.2001.01217.x

Transcriptional strength of the three PCR alleles of the α1 h1,2 enhancer and the α2 h1,2 allele in a luciferase gene reporter assay. (a) Construction: hs1,2 elements were cloned as Bam HI– Xho I fragments in Bam HI– Sal I sites of the pGL3-pVH vector. (b) Estimation of luciferase activity for the four fragments. For each assay, luciferase light units were divided by β galactosidase light units to normalise the cell transfection efficiency. Results are reported as fold-activation as compared to the pVH promoter basal activity. Mean ±SEM of four independent experiments with six assays in parallel. * P = 0·005, ** P = 0·024 and *** P = 0·019 as compared to α1A, α1B and α1C, respectively ( t -test for paired samples).
Figure Legend Snippet: Transcriptional strength of the three PCR alleles of the α1 h1,2 enhancer and the α2 h1,2 allele in a luciferase gene reporter assay. (a) Construction: hs1,2 elements were cloned as Bam HI– Xho I fragments in Bam HI– Sal I sites of the pGL3-pVH vector. (b) Estimation of luciferase activity for the four fragments. For each assay, luciferase light units were divided by β galactosidase light units to normalise the cell transfection efficiency. Results are reported as fold-activation as compared to the pVH promoter basal activity. Mean ±SEM of four independent experiments with six assays in parallel. * P = 0·005, ** P = 0·024 and *** P = 0·019 as compared to α1A, α1B and α1C, respectively ( t -test for paired samples).

Techniques Used: Polymerase Chain Reaction, Luciferase, Reporter Assay, Clone Assay, Plasmid Preparation, Activity Assay, Transfection, Activation Assay

33) Product Images from "PIAS Proteins Modulate Transcription Factors by Functioning as SUMO-1 Ligases"

Article Title: PIAS Proteins Modulate Transcription Factors by Functioning as SUMO-1 Ligases

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.22.14.5222-5234.2002

The SUMO-1-tethering region and the ligase domain are critical for the ability of PIAS proteins to coregulate AR-dependent transcription. HeLa (A) and COS-1 cells (B) cultured on 12-well plates were cotransfected with 200 ng of pARE 2 TATA-LUC, 20 ng of pCMVβ, 5 ng of pcDNA-Flag-hAR, and 10, 50, or 100 ng of pFLAG-ARIP3, pFLAG-ARIP3 (W383A), or pFLAG-ARIP3Δ467-487 and treated or not treated with 100 nM testosterone (T). After normalization for transfection efficiency by using β-galactosidase activity, reporter gene activities were expressed relative to those of AR plus T without a coregulator (set at 100). wt, wild type. (C) The same experiment as in panel B, but instead of ARIP3 expression plasmids, COS-1 cells were cotransfected with pFLAG-PIAS1, pFLAG-PIAS1(W372A), or pFLAG-PIAS1Δ310-407. (D and E) Effect of cotransfected ARIP3 on the AR mutant with the SUMO-1 attachment lysines mutated to arginines [AR(K→R)] compared to that on wild-type AR. The experimental conditions were the same as those in panels A and B. The values represent means ± standard deviations from three to six independent experiments.
Figure Legend Snippet: The SUMO-1-tethering region and the ligase domain are critical for the ability of PIAS proteins to coregulate AR-dependent transcription. HeLa (A) and COS-1 cells (B) cultured on 12-well plates were cotransfected with 200 ng of pARE 2 TATA-LUC, 20 ng of pCMVβ, 5 ng of pcDNA-Flag-hAR, and 10, 50, or 100 ng of pFLAG-ARIP3, pFLAG-ARIP3 (W383A), or pFLAG-ARIP3Δ467-487 and treated or not treated with 100 nM testosterone (T). After normalization for transfection efficiency by using β-galactosidase activity, reporter gene activities were expressed relative to those of AR plus T without a coregulator (set at 100). wt, wild type. (C) The same experiment as in panel B, but instead of ARIP3 expression plasmids, COS-1 cells were cotransfected with pFLAG-PIAS1, pFLAG-PIAS1(W372A), or pFLAG-PIAS1Δ310-407. (D and E) Effect of cotransfected ARIP3 on the AR mutant with the SUMO-1 attachment lysines mutated to arginines [AR(K→R)] compared to that on wild-type AR. The experimental conditions were the same as those in panels A and B. The values represent means ± standard deviations from three to six independent experiments.

Techniques Used: Cell Culture, Transfection, Activity Assay, Expressing, Mutagenesis

34) Product Images from "PNRC is a unique nuclear receptor coactivator that stimulates RNA polymerase III-dependent transcription"

Article Title: PNRC is a unique nuclear receptor coactivator that stimulates RNA polymerase III-dependent transcription

Journal: Journal of Molecular Signaling

doi: 10.1186/1750-2187-2-5

PNRC interacts with a subunit of RNA pol III . A , AD Gal4 -RPC39 212–316 expression plasmid was isolated from a human mammary gland expression library screening using DBD Gal4 -PNRC 270–327 as bait. To confirm the interaction and the specificity of the interaction between PNRC and RPC39, yeast strain Y187 was cotransformed with the expression plasmids for the expression of the fusion proteins as indicated, and transformants containing these plasmids were selected by growth on SD/-Leu/-Trp agar plates. The expression of interacting hybrid proteins in Y187 transformants was analyzed for Lac Z expression. DBD Gal4 (DBD) and DBD Gal4 -human lamin C (hLC) expression plasmids were included as background and negative control, respectively. β-Galactosidase activities in liquid cultures are expressed in Miller units as mean ± s.d. of three independent assays. B , PNRC interacts with the C-terminus, amino acids 212–316, of the human Pol III subunit RPC39. The yeast expression plasmids, pACT2-RPC39 and pACT2-RPC39 1–212 , for the expression of AD Gal4 -RPC39 wild type (WT) or AD Gal4 -RPC 1–212 (1–212) fusion proteins were constructed as described in 'Methods'. The expression plasmids for AD Gal4 -RPC39 212–316 (212–316) and AD Gal4 -RPC39 241–316 (241–316) were isolated from library screening through their interaction with PNRC 270–327 . Y187 cells were cotransformed with the expression plasmids coding for the fusion proteins as indicated. The selection of Y187 transformants and the β-Gal assays on transformants were performed as described in A .
Figure Legend Snippet: PNRC interacts with a subunit of RNA pol III . A , AD Gal4 -RPC39 212–316 expression plasmid was isolated from a human mammary gland expression library screening using DBD Gal4 -PNRC 270–327 as bait. To confirm the interaction and the specificity of the interaction between PNRC and RPC39, yeast strain Y187 was cotransformed with the expression plasmids for the expression of the fusion proteins as indicated, and transformants containing these plasmids were selected by growth on SD/-Leu/-Trp agar plates. The expression of interacting hybrid proteins in Y187 transformants was analyzed for Lac Z expression. DBD Gal4 (DBD) and DBD Gal4 -human lamin C (hLC) expression plasmids were included as background and negative control, respectively. β-Galactosidase activities in liquid cultures are expressed in Miller units as mean ± s.d. of three independent assays. B , PNRC interacts with the C-terminus, amino acids 212–316, of the human Pol III subunit RPC39. The yeast expression plasmids, pACT2-RPC39 and pACT2-RPC39 1–212 , for the expression of AD Gal4 -RPC39 wild type (WT) or AD Gal4 -RPC 1–212 (1–212) fusion proteins were constructed as described in 'Methods'. The expression plasmids for AD Gal4 -RPC39 212–316 (212–316) and AD Gal4 -RPC39 241–316 (241–316) were isolated from library screening through their interaction with PNRC 270–327 . Y187 cells were cotransformed with the expression plasmids coding for the fusion proteins as indicated. The selection of Y187 transformants and the β-Gal assays on transformants were performed as described in A .

Techniques Used: Expressing, Plasmid Preparation, Isolation, Library Screening, Negative Control, Construct, Selection

35) Product Images from "CIN85 modulates TGFβ signaling by promoting the presentation of TGFβ receptors on the cell surface"

Article Title: CIN85 modulates TGFβ signaling by promoting the presentation of TGFβ receptors on the cell surface

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201411025

Dominant-negative Rab11 inhibits the effect of CIN85 on TGFβ signaling. (A) HEK293T cells were transfected with HA-TβRI, Flag-CIN85, and the dominant-negative myc-Rab11(S25N) mutant, as indicated. 48 h after transfection, cell surface proteins were biotinylated and isolated by neutravidin-agarose pull-down. The amount of biotinylated HA-TβRI was analyzed by immunoblotting with HA antibodies. Cells transfected with HA-TβRI, but not treated with biotin, were used as a control for specificity of neutravidin-agarose pull-down. The levels of HA-TβRI, Flag-CIN85, and myc-Rab11(S25N) expression were determined by immunoblotting of total cell lysates. (B) HEK293T cells that had been transfected with CAGA 12 -Luc, Flag-CIN85, and myc-Rab11(S25N), as indicated, were either treated with TGFβ (5 ng/ml) for 20 h (shaded bars) or not (open bars), and the luciferase activity in the cell lysates was measured. Transfection efficiency was normalized to β-galactosidase activity. The data are presented as the mean (in percentage of control) of six independent experiments. Error bars are SD. *, P
Figure Legend Snippet: Dominant-negative Rab11 inhibits the effect of CIN85 on TGFβ signaling. (A) HEK293T cells were transfected with HA-TβRI, Flag-CIN85, and the dominant-negative myc-Rab11(S25N) mutant, as indicated. 48 h after transfection, cell surface proteins were biotinylated and isolated by neutravidin-agarose pull-down. The amount of biotinylated HA-TβRI was analyzed by immunoblotting with HA antibodies. Cells transfected with HA-TβRI, but not treated with biotin, were used as a control for specificity of neutravidin-agarose pull-down. The levels of HA-TβRI, Flag-CIN85, and myc-Rab11(S25N) expression were determined by immunoblotting of total cell lysates. (B) HEK293T cells that had been transfected with CAGA 12 -Luc, Flag-CIN85, and myc-Rab11(S25N), as indicated, were either treated with TGFβ (5 ng/ml) for 20 h (shaded bars) or not (open bars), and the luciferase activity in the cell lysates was measured. Transfection efficiency was normalized to β-galactosidase activity. The data are presented as the mean (in percentage of control) of six independent experiments. Error bars are SD. *, P

Techniques Used: Dominant Negative Mutation, Transfection, Mutagenesis, Isolation, Expressing, Luciferase, Activity Assay

CIN85 enhances TGFβ transcriptional activity. (A) HEK293T cells, transfected with CAGA 12 -Luc (left) or xFAST/ARE-Luc (right) and different amounts of Flag-CIN85 plasmid, were not treated (open bars) or treated with 5 ng/ml TGFβ (shaded bars) for 20 h, and luciferase activity was measured. The results are presented as the mean (in percentage of control) of six independent experiments. Error bars represent SD. (B) qRT-PCR analysis for expression for PAI-1 was performed on mRNA extracted from PC-3U cells transiently transfected with empty pCDNA3 vector or Flag-CIN85 and treated with 5 ng/ml TGFβ for the indicated time periods. The results were normalized on the basis of GAPDH mRNA expression. The data are plotted as the mean fold induction of TGFβ-stimulated mRNA levels relative to unstimulated levels (0 h set to 1) with SD determined from triplicate measurements. Results of one representative experiment out of three performed are shown. (C) HEK293T cells transfected with CAGA 12 -Luc reporter and control plasmid (open bars) or Flag-CIN85 plasmid (shaded bars). The cells were pretreated for 1 h with DMSO or TβRI kinase inhibitor SB505124 (10 µM) and then incubated with 5 ng/ml TGFβ for 20 h as indicated. Luciferase activity was measured and transfection efficiency was normalized to β-galactosidase activity. The results are presented as the mean (in percentage of control) of three independent experiments. Error bars represent SD. *, P
Figure Legend Snippet: CIN85 enhances TGFβ transcriptional activity. (A) HEK293T cells, transfected with CAGA 12 -Luc (left) or xFAST/ARE-Luc (right) and different amounts of Flag-CIN85 plasmid, were not treated (open bars) or treated with 5 ng/ml TGFβ (shaded bars) for 20 h, and luciferase activity was measured. The results are presented as the mean (in percentage of control) of six independent experiments. Error bars represent SD. (B) qRT-PCR analysis for expression for PAI-1 was performed on mRNA extracted from PC-3U cells transiently transfected with empty pCDNA3 vector or Flag-CIN85 and treated with 5 ng/ml TGFβ for the indicated time periods. The results were normalized on the basis of GAPDH mRNA expression. The data are plotted as the mean fold induction of TGFβ-stimulated mRNA levels relative to unstimulated levels (0 h set to 1) with SD determined from triplicate measurements. Results of one representative experiment out of three performed are shown. (C) HEK293T cells transfected with CAGA 12 -Luc reporter and control plasmid (open bars) or Flag-CIN85 plasmid (shaded bars). The cells were pretreated for 1 h with DMSO or TβRI kinase inhibitor SB505124 (10 µM) and then incubated with 5 ng/ml TGFβ for 20 h as indicated. Luciferase activity was measured and transfection efficiency was normalized to β-galactosidase activity. The results are presented as the mean (in percentage of control) of three independent experiments. Error bars represent SD. *, P

Techniques Used: Activity Assay, Transfection, Plasmid Preparation, Luciferase, Quantitative RT-PCR, Expressing, Incubation

CIN85 interacts with TGFβ receptors. (A) HEK293T cells were transfected with Flag-CIN85 in the absence or presence of HA-TβRI and incubated with 5 ng/ml TGFβ for 15 min, as indicated. The cell lysates were subjected to immunoprecipitation with HA antibodies followed by blotting with Flag antibodies. The levels of HA-TβRI and Flag-CIN85 expression were determined by immunoblotting of total cell lysates. (B) PC-3U cells were treated with 5 ng/ml TGFβ for 15 min and endogenous TβRI (left) or CIN85 (right) were immunoprecipitated with 1 µg of goat anti-TβRI or rabbit anti-CIN85 antibodies, respectively. Immunoprecipitates were analyzed by immunoblotting for the presence of CIN85 or TβRI, as indicated. Total cell lysate was also subjected to immunoblotting for CIN85 and TβRI, and for phosphorylated Smad2 to determine activation of TGFβ signaling (bottom). (C) HEK293T cells were transfected with Flag-CIN85 in the absence or presence of His-TβRII and incubated with 5 ng/ml TGFβ for 15 min, as indicated. TβRII was immunoprecipitated from the cell lysates with His antibodies. Coimmunoprecipitated CIN85 and immunoprecipitated TβRII were detected by immunoblotting with Flag and His antibodies, respectively. The levels of His-TβRII and Flag-CIN85 expression were determined by immunoblotting of total cell lysates. (D) Schematic illustration of the CIN85 molecule. The full-length molecule (FL) and the parts of CIN85 included into the deletion mutants containing three SH3 domains (3SH3) or proline-rich and coiled-coil domains (PcC) are indicated. (E) TβRI interacts with the N-terminal part of CIN85. HA-TβRI was transfected into HEK293T cells together with full-length Flag-CIN85 or with deletion mutants including three SH3 domains (3SH3) or proline-rich and coiled coil domains (PcC). The cells were incubated with 5 ng/ml TGFβ for 15 min and TβRI was immunoprecipitated from the cell lysates with HA antibodies. The coimmunoprecipitated CIN85 or CIN85 deletion mutants were detected by immunoblotting with Flag antibodies. The expression of HA-TβRI and Flag-CIN85 molecules was determined by immunoblotting of total cell lysates. (F) TβRII interacts with the N-terminal part of CIN85. HA-TβRII was transfected into HEK293T cells together with full-length Flag-CIN85 or with its deletion mutants. The cells were treated as in E and TβRII was immunoprecipitated from the cell lysates with HA antibodies. The coimmunoprecipitated CIN85 or CIN85 deletion mutants were detected by immunoblotting with Flag antibodies. (G) The transcriptional activity of TGFβ is enhanced by full-length CIN85 but not by its N- or C-terminal fragments. HEK293T cells, transfected with CAGA 12 -Luc and full-length CIN85 or its N- or C-terminal parts, were treated (shaded bars) or not (open bars) with TGFβ (5 ng/ml) for 20 h. Luciferase activity was measured and transfection efficiency was normalized to β-galactosidase activity. The results are presented as the mean (in percentage of control) of three independent experiments. Error bars are SD. *, P
Figure Legend Snippet: CIN85 interacts with TGFβ receptors. (A) HEK293T cells were transfected with Flag-CIN85 in the absence or presence of HA-TβRI and incubated with 5 ng/ml TGFβ for 15 min, as indicated. The cell lysates were subjected to immunoprecipitation with HA antibodies followed by blotting with Flag antibodies. The levels of HA-TβRI and Flag-CIN85 expression were determined by immunoblotting of total cell lysates. (B) PC-3U cells were treated with 5 ng/ml TGFβ for 15 min and endogenous TβRI (left) or CIN85 (right) were immunoprecipitated with 1 µg of goat anti-TβRI or rabbit anti-CIN85 antibodies, respectively. Immunoprecipitates were analyzed by immunoblotting for the presence of CIN85 or TβRI, as indicated. Total cell lysate was also subjected to immunoblotting for CIN85 and TβRI, and for phosphorylated Smad2 to determine activation of TGFβ signaling (bottom). (C) HEK293T cells were transfected with Flag-CIN85 in the absence or presence of His-TβRII and incubated with 5 ng/ml TGFβ for 15 min, as indicated. TβRII was immunoprecipitated from the cell lysates with His antibodies. Coimmunoprecipitated CIN85 and immunoprecipitated TβRII were detected by immunoblotting with Flag and His antibodies, respectively. The levels of His-TβRII and Flag-CIN85 expression were determined by immunoblotting of total cell lysates. (D) Schematic illustration of the CIN85 molecule. The full-length molecule (FL) and the parts of CIN85 included into the deletion mutants containing three SH3 domains (3SH3) or proline-rich and coiled-coil domains (PcC) are indicated. (E) TβRI interacts with the N-terminal part of CIN85. HA-TβRI was transfected into HEK293T cells together with full-length Flag-CIN85 or with deletion mutants including three SH3 domains (3SH3) or proline-rich and coiled coil domains (PcC). The cells were incubated with 5 ng/ml TGFβ for 15 min and TβRI was immunoprecipitated from the cell lysates with HA antibodies. The coimmunoprecipitated CIN85 or CIN85 deletion mutants were detected by immunoblotting with Flag antibodies. The expression of HA-TβRI and Flag-CIN85 molecules was determined by immunoblotting of total cell lysates. (F) TβRII interacts with the N-terminal part of CIN85. HA-TβRII was transfected into HEK293T cells together with full-length Flag-CIN85 or with its deletion mutants. The cells were treated as in E and TβRII was immunoprecipitated from the cell lysates with HA antibodies. The coimmunoprecipitated CIN85 or CIN85 deletion mutants were detected by immunoblotting with Flag antibodies. (G) The transcriptional activity of TGFβ is enhanced by full-length CIN85 but not by its N- or C-terminal fragments. HEK293T cells, transfected with CAGA 12 -Luc and full-length CIN85 or its N- or C-terminal parts, were treated (shaded bars) or not (open bars) with TGFβ (5 ng/ml) for 20 h. Luciferase activity was measured and transfection efficiency was normalized to β-galactosidase activity. The results are presented as the mean (in percentage of control) of three independent experiments. Error bars are SD. *, P

Techniques Used: Transfection, Incubation, Immunoprecipitation, Expressing, Activation Assay, Periodic Counter-current Chromatography, Activity Assay, Luciferase

36) Product Images from "Ets-1 as an early response gene against hypoxia-induced apoptosis in pancreatic β-cells"

Article Title: Ets-1 as an early response gene against hypoxia-induced apoptosis in pancreatic β-cells

Journal: Cell Death & Disease

doi: 10.1038/cddis.2015.8

Severe hypoxia-induced degradation of Ets-1 is correlated to its transactivation activity. ( a ) Effect of oxygen concentration on the protein level of Ets-1. MIN6 cells were exposed to 2, 4, 6, 8 and 10% O 2 for 1 h. Total proteins were extracted and analyzed by western blotting. ( b ) Effect of oxygen concentration on the transactivation activity of Ets-1. MIN6 cells were transfected with pEGFP-Ets-1 WT . At 24 h following transfection, cells were exposed to 2, 4, 6, 8 and 10% O 2 for 1 h, followed by RNA extraction and qRT-PCR analysis. The average values and standard deviations ( n =3) are shown. *and **indicate P
Figure Legend Snippet: Severe hypoxia-induced degradation of Ets-1 is correlated to its transactivation activity. ( a ) Effect of oxygen concentration on the protein level of Ets-1. MIN6 cells were exposed to 2, 4, 6, 8 and 10% O 2 for 1 h. Total proteins were extracted and analyzed by western blotting. ( b ) Effect of oxygen concentration on the transactivation activity of Ets-1. MIN6 cells were transfected with pEGFP-Ets-1 WT . At 24 h following transfection, cells were exposed to 2, 4, 6, 8 and 10% O 2 for 1 h, followed by RNA extraction and qRT-PCR analysis. The average values and standard deviations ( n =3) are shown. *and **indicate P

Techniques Used: Activity Assay, Concentration Assay, Western Blot, Transfection, RNA Extraction, Quantitative RT-PCR

37) Product Images from "Tumor Necrosis Factor Alpha Transcription in Macrophages Is Attenuated by an Autocrine Factor That Preferentially Induces NF-?B p50"

Article Title: Tumor Necrosis Factor Alpha Transcription in Macrophages Is Attenuated by an Autocrine Factor That Preferentially Induces NF-?B p50

Journal: Molecular and Cellular Biology

doi:

The distal TNF-α κB sites are poorly activated by p65. Reporter plasmids (1 μg) containing four tandem copies of the indicated κB sites upstream of a minimal promoter reporter gene (TK-Luc) were cotransfected into ANA-1 cells with various ratios of p50 and p65 expression vectors (1 μg in total) and an internal standard, pRSV-βgal (0.5 μg). After 2 days, the cells were induced with LPS for 4 h and harvested, and the lysates were assayed for luciferase and β-galactosidase activities. Luciferase activity was normalized to β-galactosidase activity for each sample. The data are from a representative experiment; similar results were obtained in two independent experiments.
Figure Legend Snippet: The distal TNF-α κB sites are poorly activated by p65. Reporter plasmids (1 μg) containing four tandem copies of the indicated κB sites upstream of a minimal promoter reporter gene (TK-Luc) were cotransfected into ANA-1 cells with various ratios of p50 and p65 expression vectors (1 μg in total) and an internal standard, pRSV-βgal (0.5 μg). After 2 days, the cells were induced with LPS for 4 h and harvested, and the lysates were assayed for luciferase and β-galactosidase activities. Luciferase activity was normalized to β-galactosidase activity for each sample. The data are from a representative experiment; similar results were obtained in two independent experiments.

Techniques Used: Expressing, Luciferase, Activity Assay

CM suppresses LPS-induced transcription from the TNF-α promoter in transfected macrophages. (A) ANA-1 macrophages were transfected with the TNF-Luc reporter plasmid (1 μg/2 × 10 6 cells), treated with LPS − CM or control CM for 16 h, and then stimulated with LPS over a 12-h time course. Relative luciferase expression was calculated by normalizing to luciferase activity in control cells at 0 h. The data represent the average of three independent experiments. (B) The same experiment was performed with a control reporter plasmid, pRSV-βgal. Relative β-galactosidase (β-gal) expression was calculated as described above for luciferase activity.
Figure Legend Snippet: CM suppresses LPS-induced transcription from the TNF-α promoter in transfected macrophages. (A) ANA-1 macrophages were transfected with the TNF-Luc reporter plasmid (1 μg/2 × 10 6 cells), treated with LPS − CM or control CM for 16 h, and then stimulated with LPS over a 12-h time course. Relative luciferase expression was calculated by normalizing to luciferase activity in control cells at 0 h. The data represent the average of three independent experiments. (B) The same experiment was performed with a control reporter plasmid, pRSV-βgal. Relative β-galactosidase (β-gal) expression was calculated as described above for luciferase activity.

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

38) Product Images from "HIF2α signaling inhibits adherens junctional disruption in acute lung injury"

Article Title: HIF2α signaling inhibits adherens junctional disruption in acute lung injury

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI77701

HIF2α induces VE-PTP expression and enhances the integrity of endothelial AJs. ( A ) HLMVECs were exposed to varying concentrations of O 2 for 8 hours. Expression of HIF1α and HIF2α was assessed by WB. ( B ) Representation of the VEPTP promoter region. HREs are shown by circled numbers, and their sequences are displayed. ( C ) HLMVECs were exposed to normoxia or 1% O 2 for 8 hours. A ChIP assay was performed to amplify the VEPTP and VE-cadherin promoters. ( D ) 293T cells were transfected with an HIF2α-DPA expression plasmid containing luciferase reporter constructs. Luciferase values were normalized to β-gal values. A schematic representation of corresponding deletion constructs is presented in the right panel. ( E ) HLMVECs infected with lentiviral HIF2α siRNA/shRNA were exposed to normoxia or 1% O 2 . Expression of VE-cadherin, VE-PTP, and HIF2α was assessed by WB. ( F - G ) Quantification of VE-PTP ( F ) and VE-cadherin ( G ) protein levels. ( H ) HLMVECs were exposed to normoxia or 1% O 2 . Expression of VEPTP and VE-cadherin at the mRNA level was assessed by quantitative PCR. ( I ) AJ integrity of HLMVECs was examined by VE-cadherin immunostaining using confocal microscopy. Scale bars: 20 μm. n = 3/group ( F – H ). Blot images were derived from samples run on parallel gels. * P
Figure Legend Snippet: HIF2α induces VE-PTP expression and enhances the integrity of endothelial AJs. ( A ) HLMVECs were exposed to varying concentrations of O 2 for 8 hours. Expression of HIF1α and HIF2α was assessed by WB. ( B ) Representation of the VEPTP promoter region. HREs are shown by circled numbers, and their sequences are displayed. ( C ) HLMVECs were exposed to normoxia or 1% O 2 for 8 hours. A ChIP assay was performed to amplify the VEPTP and VE-cadherin promoters. ( D ) 293T cells were transfected with an HIF2α-DPA expression plasmid containing luciferase reporter constructs. Luciferase values were normalized to β-gal values. A schematic representation of corresponding deletion constructs is presented in the right panel. ( E ) HLMVECs infected with lentiviral HIF2α siRNA/shRNA were exposed to normoxia or 1% O 2 . Expression of VE-cadherin, VE-PTP, and HIF2α was assessed by WB. ( F - G ) Quantification of VE-PTP ( F ) and VE-cadherin ( G ) protein levels. ( H ) HLMVECs were exposed to normoxia or 1% O 2 . Expression of VEPTP and VE-cadherin at the mRNA level was assessed by quantitative PCR. ( I ) AJ integrity of HLMVECs was examined by VE-cadherin immunostaining using confocal microscopy. Scale bars: 20 μm. n = 3/group ( F – H ). Blot images were derived from samples run on parallel gels. * P

Techniques Used: Expressing, Western Blot, Chromatin Immunoprecipitation, Transfection, Plasmid Preparation, Luciferase, Construct, Infection, shRNA, Real-time Polymerase Chain Reaction, Immunostaining, Confocal Microscopy, Derivative Assay

39) Product Images from "p38 Regulates Expression of Osteoblast-specific Genes by Phosphorylation of Osterix *"

Article Title: p38 Regulates Expression of Osteoblast-specific Genes by Phosphorylation of Osterix *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.123612

Fmod and Ibsp transcriptional activation by Osx and BMP-2. A , cells were co-transfected with 2-kb pFmod-Luc reporter vector and either mock or Osx construct overnight and treated with or without BMP-2 in medium without serum for 24 h. B , cells were transfected with 2-kb pFmod-Luc or pFmod300-Luc reporter vectors overnight and treated with BMP-2 in medium without serum for 24 h. C , cells were co-transfected with pIbsp-Luc reporter vector and either mock or Osx construct overnight and treated with or without BMP-2 in medium without serum for 24 h. A , B , and C , luciferase activity was measured and normalized against β-galactosidase activity. Relative luciferase activities were expressed as mean ± S.E. for triplicates from five independent experiments (*, p
Figure Legend Snippet: Fmod and Ibsp transcriptional activation by Osx and BMP-2. A , cells were co-transfected with 2-kb pFmod-Luc reporter vector and either mock or Osx construct overnight and treated with or without BMP-2 in medium without serum for 24 h. B , cells were transfected with 2-kb pFmod-Luc or pFmod300-Luc reporter vectors overnight and treated with BMP-2 in medium without serum for 24 h. C , cells were co-transfected with pIbsp-Luc reporter vector and either mock or Osx construct overnight and treated with or without BMP-2 in medium without serum for 24 h. A , B , and C , luciferase activity was measured and normalized against β-galactosidase activity. Relative luciferase activities were expressed as mean ± S.E. for triplicates from five independent experiments (*, p

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

40) Product Images from "Combinatorial regulation of endothelial gene expression by ets and forkhead transcription factors"

Article Title: Combinatorial regulation of endothelial gene expression by ets and forkhead transcription factors

Journal: Cell

doi: 10.1016/j.cell.2008.10.049

Identification of a 44-bp Mef2c endothelial-specific enhancer (A) A schematic representation of the mouse Mef2c locus is shown on the top line with exons depicted as vertical lines. The red boxes denotes the sizes and positions of the F7 and F10 fragments. F10 contains three evolutionarily conserved regions, denoted CR1-3. The lower portion of (A) depicts the deletion constructs of Mef2c F10. CR3 contains a neural crest specific enhancer. CR2 contains an endothelial specific enhancer, which encompasses a 44-bp deeply conserved region that is sufficient for endothelial enhancer activity in vivo . Endothelial and neural crest activity of each of the deletion constructs is denoted at the right as a + or -. The total number of transgenic embryos and the number that directed β-galactosidase expression to either the neural crest or endothelium are denoted at the far right of (A). (B-G) Representative X-gal stained transgenic embryos for each of the Mef2c F10 transgene deletion constructs depicted in (A). (H-M) Expression of the Mef2c F10-44- lacZ construct is specific to endothelial cells from blood island (bl) stage at E7.5 (H) throughout early endothelial development at E8.0 (I) and E8.5 (J, K). Transverse sections through an X-gal stained E9.5 transgenic embryo (L, M) demonstrate that transgene expression is restricted to endothelial cells throughout the vasculature, including the endocardium (end). al, allantois; BAA, branchial arch artery; CV, cardinal vein; DA, dorsal aorta; DRG, dorsal root ganglia; ec, ectoplacental cone; hrt, heart; LV, left ventricle; NC, neural crest; NT, neural tube; RV, right ventricle; SV, sinus venosus; YS, yolk sac.
Figure Legend Snippet: Identification of a 44-bp Mef2c endothelial-specific enhancer (A) A schematic representation of the mouse Mef2c locus is shown on the top line with exons depicted as vertical lines. The red boxes denotes the sizes and positions of the F7 and F10 fragments. F10 contains three evolutionarily conserved regions, denoted CR1-3. The lower portion of (A) depicts the deletion constructs of Mef2c F10. CR3 contains a neural crest specific enhancer. CR2 contains an endothelial specific enhancer, which encompasses a 44-bp deeply conserved region that is sufficient for endothelial enhancer activity in vivo . Endothelial and neural crest activity of each of the deletion constructs is denoted at the right as a + or -. The total number of transgenic embryos and the number that directed β-galactosidase expression to either the neural crest or endothelium are denoted at the far right of (A). (B-G) Representative X-gal stained transgenic embryos for each of the Mef2c F10 transgene deletion constructs depicted in (A). (H-M) Expression of the Mef2c F10-44- lacZ construct is specific to endothelial cells from blood island (bl) stage at E7.5 (H) throughout early endothelial development at E8.0 (I) and E8.5 (J, K). Transverse sections through an X-gal stained E9.5 transgenic embryo (L, M) demonstrate that transgene expression is restricted to endothelial cells throughout the vasculature, including the endocardium (end). al, allantois; BAA, branchial arch artery; CV, cardinal vein; DA, dorsal aorta; DRG, dorsal root ganglia; ec, ectoplacental cone; hrt, heart; LV, left ventricle; NC, neural crest; NT, neural tube; RV, right ventricle; SV, sinus venosus; YS, yolk sac.

Techniques Used: Construct, Activity Assay, In Vivo, Transgenic Assay, Expressing, Staining

Related Articles

Activity Assay:

Article Title: Natural antisense RNA inhibits the expression of BCMA, a tumour necrosis factor receptor homologue.
Article Snippet: .. The luciferase activity was measured using the Reporter assay system (Promega, Madison, WI). β-galactosidase activity was measured using the Luminescent β-galactosidase reporter system (Clontech, Palo Alto, CA). ..

Article Title: Hypoxic Preconditioning Enhances the Benefit of Cardiac Progenitor-Cell Therapy for Treatment of Myocardial Infarction by Inducing CXCR4 Expression
Article Snippet: .. Cardiac protein extracts were prepared and β-galactosidase activity was assayed by using the Luminescent beta-galactosidase Reporter System 3 (Clontech) and a Monolight 3010 (Pharmingen) as directed by the manufacturer’s protocol. .. One month after surgically induced MI and intravenous injection of CLK-LacZ cells, mouse hearts were harvested, embedded in OCT compound, snap-frozen, cut into 5-μm sections, and double-immunostained with FITC-conjugated anti-lacZ antibody (1:500, [Abcam Inc.]) and with anti-cTnI (1:100, [Santa Cruz Biotechnology, Inc.]), anti-vW Factor (1:100,Sigma), or anti-SMA (1:100, Zymed Lab Inc., San Francisco, CA) antibodies.

Article Title: High-Level Replication of Human Immunodeficiency Virus in Thymocytes Requires NF-?B Activation through Interaction with Thymic Epithelial Cells
Article Snippet: .. Luciferase activity was measured by standard procedures, as previously described , and β-galactosidase activity was measured by using the luminescent β-galactosidase genetic reporter system II (Clontech, Palo Alto, Calif.). .. Normalized luciferase activity represents the ratio of luciferase to β-galactosidase activities.

Article Title: Tumor Necrosis Factor Alpha Transcription in Macrophages Is Attenuated by an Autocrine Factor That Preferentially Induces NF-?B p50
Article Snippet: .. Forty hours after transfection, the cells were treated with LPS for the indicated time periods, lysed, and analyzed for luciferase activity by using an Enhanced Luciferase Assay kit (Analytical Luminescent Laboratory) or, where appropriate, for both luciferase and β-galactosidase activities by using the Luminescent β-Galactosidase Genetic Reporter System II (Clontech). .. In experiments lacking the β-galactosidase control, the protein concentration of each lysate was measured (Bio-Rad) and used to normalize luciferase activity.

Article Title: ?-Adrenergic receptor-induced activation of nerve growth factor gene transcription in rat cerebral cortex involves CCAAT/enhancer-binding protein ?
Article Snippet: .. Cells were harvested after 48 hr, and lysates were analyzed for luciferase activity by using the Enhanced Luciferase Assay Kit (Analytical Luminescence Laboratory, San Diego) and for β-galactosidase activity by using the Luminescent β-galactosidase Genetic Reporter System II (CLONTECH). .. When β-galactosidase was not used, protein concentration in each lysate was measured (Bio-Rad) to normalize luciferase activity.

Article Title: Interleukin-6-Specific Activation of the C/EBP? Gene in Hepatocytes Is Mediated by Stat3 and Sp1
Article Snippet: .. Background reading was determined by measuring cell lysate from mock-transfected cells from two independent dishes. β-Galactosidase activity, which was used as an internal standard for transfection efficiency, was assayed according to the protocol for Luminescent β-galactosidase Genetic Reporter System II (Clontech Laboratories). ..

Transfection:

Article Title: Tumor Necrosis Factor Alpha Transcription in Macrophages Is Attenuated by an Autocrine Factor That Preferentially Induces NF-?B p50
Article Snippet: .. Forty hours after transfection, the cells were treated with LPS for the indicated time periods, lysed, and analyzed for luciferase activity by using an Enhanced Luciferase Assay kit (Analytical Luminescent Laboratory) or, where appropriate, for both luciferase and β-galactosidase activities by using the Luminescent β-Galactosidase Genetic Reporter System II (Clontech). .. In experiments lacking the β-galactosidase control, the protein concentration of each lysate was measured (Bio-Rad) and used to normalize luciferase activity.

Article Title: Interleukin-6-Specific Activation of the C/EBP? Gene in Hepatocytes Is Mediated by Stat3 and Sp1
Article Snippet: .. Background reading was determined by measuring cell lysate from mock-transfected cells from two independent dishes. β-Galactosidase activity, which was used as an internal standard for transfection efficiency, was assayed according to the protocol for Luminescent β-galactosidase Genetic Reporter System II (Clontech Laboratories). ..

Luciferase:

Article Title: Natural antisense RNA inhibits the expression of BCMA, a tumour necrosis factor receptor homologue.
Article Snippet: .. The luciferase activity was measured using the Reporter assay system (Promega, Madison, WI). β-galactosidase activity was measured using the Luminescent β-galactosidase reporter system (Clontech, Palo Alto, CA). ..

Article Title: High-Level Replication of Human Immunodeficiency Virus in Thymocytes Requires NF-?B Activation through Interaction with Thymic Epithelial Cells
Article Snippet: .. Luciferase activity was measured by standard procedures, as previously described , and β-galactosidase activity was measured by using the luminescent β-galactosidase genetic reporter system II (Clontech, Palo Alto, Calif.). .. Normalized luciferase activity represents the ratio of luciferase to β-galactosidase activities.

Article Title: Tumor Necrosis Factor Alpha Transcription in Macrophages Is Attenuated by an Autocrine Factor That Preferentially Induces NF-?B p50
Article Snippet: .. Forty hours after transfection, the cells were treated with LPS for the indicated time periods, lysed, and analyzed for luciferase activity by using an Enhanced Luciferase Assay kit (Analytical Luminescent Laboratory) or, where appropriate, for both luciferase and β-galactosidase activities by using the Luminescent β-Galactosidase Genetic Reporter System II (Clontech). .. In experiments lacking the β-galactosidase control, the protein concentration of each lysate was measured (Bio-Rad) and used to normalize luciferase activity.

Article Title: ?-Adrenergic receptor-induced activation of nerve growth factor gene transcription in rat cerebral cortex involves CCAAT/enhancer-binding protein ?
Article Snippet: .. Cells were harvested after 48 hr, and lysates were analyzed for luciferase activity by using the Enhanced Luciferase Assay Kit (Analytical Luminescence Laboratory, San Diego) and for β-galactosidase activity by using the Luminescent β-galactosidase Genetic Reporter System II (CLONTECH). .. When β-galactosidase was not used, protein concentration in each lysate was measured (Bio-Rad) to normalize luciferase activity.

Reporter Assay:

Article Title: Natural antisense RNA inhibits the expression of BCMA, a tumour necrosis factor receptor homologue.
Article Snippet: .. The luciferase activity was measured using the Reporter assay system (Promega, Madison, WI). β-galactosidase activity was measured using the Luminescent β-galactosidase reporter system (Clontech, Palo Alto, CA). ..

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    TaKaRa β galactosidase reporter plasmid
    Effects of expression of JEV NS proteins on reporter activity in BHK-21 cells. BHK-21 cells were cotransfected with a reporter gene and plasmids expressing various JEV NS proteins. The reporters include a luciferase gene (A and B) and a <t>β-galactosidase</t> gene (C and D). At 24 h (A and B) or 48 h (C and D) posttransfection, the cells were lysed and assayed for their luciferase or β-galactosidase activities. 2A-R and 4A-R in panels B and D are the constructs with inserts in opposite orientation.
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    Effects of expression of JEV NS proteins on reporter activity in BHK-21 cells. BHK-21 cells were cotransfected with a reporter gene and plasmids expressing various JEV NS proteins. The reporters include a luciferase gene (A and B) and a β-galactosidase gene (C and D). At 24 h (A and B) or 48 h (C and D) posttransfection, the cells were lysed and assayed for their luciferase or β-galactosidase activities. 2A-R and 4A-R in panels B and D are the constructs with inserts in opposite orientation.

    Journal: Journal of Virology

    Article Title: Membrane Permeabilization by Small Hydrophobic Nonstructural Proteins of Japanese Encephalitis Virus

    doi:

    Figure Lengend Snippet: Effects of expression of JEV NS proteins on reporter activity in BHK-21 cells. BHK-21 cells were cotransfected with a reporter gene and plasmids expressing various JEV NS proteins. The reporters include a luciferase gene (A and B) and a β-galactosidase gene (C and D). At 24 h (A and B) or 48 h (C and D) posttransfection, the cells were lysed and assayed for their luciferase or β-galactosidase activities. 2A-R and 4A-R in panels B and D are the constructs with inserts in opposite orientation.

    Article Snippet: By using Lipofectamine PLUS (GIBCO-BRL) as described by the manufacturer, BHK-21 cells in a six-well plate were cotransfected with β-galactosidase reporter plasmid, pCMVβ (Clontech) (0.5 μg per well) and each of the plasmids expressing the various JEV proteins (2.5 μg per well).

    Techniques: Expressing, Activity Assay, Luciferase, Construct