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TaKaRa pg5 e1b cat
REP binds hTBP in vivo as determined by GAL4 two-hybrid system. All of the negative controls resulted in relatively low signals <t>(pG5</t> [reporter plasmid pG5 <t>E1b</t> Luc] + pM [GAL4 DNA binding domain-based expression plasmid] + pVP16 [HSV VP16 transactivation domain-based expression plasmid], pG5 + pMhTBP + pVP16, pG5 + pM + pVP16rep or pG5). In contrast, both the positive control (pG5 + pM53 [p53]; + pVP16-T [large T]) and pG5 + PMhTBP + pVP16rep gave significantly higher signals. Furthermore, pVP16 RepΔEco+pMhTBP showed higher luciferase activity than pVP16 Rep+pMhTBP. On the other hand, pVP RepΔMlu showed much higher activity than either pVP16 Rep or pVP16 RepΔEco. Thus, these data are consistent with a significant REP-TBP interaction in vivo. Data (means + standard deviations [error bars] of the relative luciferase activity) were calculated from triplicate cultures and are representative of three independent experiments.
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Images

1) Product Images from "Expression of Human Herpesvirus 6B rep within Infected Cells and Binding of Its Gene Product to the TATA-Binding Protein In Vitro and In Vivo"

Article Title: Expression of Human Herpesvirus 6B rep within Infected Cells and Binding of Its Gene Product to the TATA-Binding Protein In Vitro and In Vivo

Journal: Journal of Virology

doi:

REP binds hTBP in vivo as determined by GAL4 two-hybrid system. All of the negative controls resulted in relatively low signals (pG5 [reporter plasmid pG5 E1b Luc] + pM [GAL4 DNA binding domain-based expression plasmid] + pVP16 [HSV VP16 transactivation domain-based expression plasmid], pG5 + pMhTBP + pVP16, pG5 + pM + pVP16rep or pG5). In contrast, both the positive control (pG5 + pM53 [p53]; + pVP16-T [large T]) and pG5 + PMhTBP + pVP16rep gave significantly higher signals. Furthermore, pVP16 RepΔEco+pMhTBP showed higher luciferase activity than pVP16 Rep+pMhTBP. On the other hand, pVP RepΔMlu showed much higher activity than either pVP16 Rep or pVP16 RepΔEco. Thus, these data are consistent with a significant REP-TBP interaction in vivo. Data (means + standard deviations [error bars] of the relative luciferase activity) were calculated from triplicate cultures and are representative of three independent experiments.
Figure Legend Snippet: REP binds hTBP in vivo as determined by GAL4 two-hybrid system. All of the negative controls resulted in relatively low signals (pG5 [reporter plasmid pG5 E1b Luc] + pM [GAL4 DNA binding domain-based expression plasmid] + pVP16 [HSV VP16 transactivation domain-based expression plasmid], pG5 + pMhTBP + pVP16, pG5 + pM + pVP16rep or pG5). In contrast, both the positive control (pG5 + pM53 [p53]; + pVP16-T [large T]) and pG5 + PMhTBP + pVP16rep gave significantly higher signals. Furthermore, pVP16 RepΔEco+pMhTBP showed higher luciferase activity than pVP16 Rep+pMhTBP. On the other hand, pVP RepΔMlu showed much higher activity than either pVP16 Rep or pVP16 RepΔEco. Thus, these data are consistent with a significant REP-TBP interaction in vivo. Data (means + standard deviations [error bars] of the relative luciferase activity) were calculated from triplicate cultures and are representative of three independent experiments.

Techniques Used: In Vivo, Plasmid Preparation, Binding Assay, Expressing, Positive Control, Luciferase, Activity Assay

2) Product Images from "Activation of Androgen Receptor Function by a Novel Nuclear Protein Kinase"

Article Title: Activation of Androgen Receptor Function by a Novel Nuclear Protein Kinase

Journal: Molecular Biology of the Cell

doi:

ANPK enhances androgen-induced transcriptional activation. (A) CV-1 cells were transfected using the calcium phosphate method with 5 μg of pPB(-285/+32)-LUC reporter plasmid along with 0.5 μg of pSG5-rAR and indicated amounts (μg) of pFLAG-ANPK(159–1191) or kinase-defective pFLAG-ANPK(K226R) in the presence or absence of 100 nM testosterone (T) as depicted. Total amount of DNA was kept constant by adding empty pFLAG-CMV-2 expression vector as needed. β-Gal expression plasmid, pCMVβ (2 μg/10-cm plate), was used to control for transfection efficiency. Luciferase (LUC) activities were normalized using β-gal activity. LUC activities are expressed relative to that of pSG5-rAR in the presence of testosterone (= 100), and the mean ± SE values of at least six independent experiments are given. (B and C) ANPK does not modulate PR- and GR-dependent transcription. (B) CV-1 cells were transfected with 5 μg of pARE 2 -E1b-CAT reporter containing two copies of the GRE/PRE/ARE motif of the rat tyrosine aminotransferase gene upstream of the adenovirus E1b TATA sequence along with 0.5 μg of pSG5-hGR, 5 μg of empty expression vector (pFLAG-CMV-2) (open bar) or pFLAG-ANPK(159–1191) (solid bar), and 2 μg of pCMVβ in the presence or absence of 100 nM dexamethasone (D). (C) CV-1 cells were transfected as in panel B, but using 0.5 μg of pSG5-hPR1 instead of pSG5-hGR in the presence or absence of 100 nM progesterone (P). CAT activities are normalized to β-gal activity and expressed relative to those achieved with pSG5-hGR or pSG5-hPR1 in the presence of P or D, respectively (= 100), and the mean ± SE values of at least three independent experiments are shown.
Figure Legend Snippet: ANPK enhances androgen-induced transcriptional activation. (A) CV-1 cells were transfected using the calcium phosphate method with 5 μg of pPB(-285/+32)-LUC reporter plasmid along with 0.5 μg of pSG5-rAR and indicated amounts (μg) of pFLAG-ANPK(159–1191) or kinase-defective pFLAG-ANPK(K226R) in the presence or absence of 100 nM testosterone (T) as depicted. Total amount of DNA was kept constant by adding empty pFLAG-CMV-2 expression vector as needed. β-Gal expression plasmid, pCMVβ (2 μg/10-cm plate), was used to control for transfection efficiency. Luciferase (LUC) activities were normalized using β-gal activity. LUC activities are expressed relative to that of pSG5-rAR in the presence of testosterone (= 100), and the mean ± SE values of at least six independent experiments are given. (B and C) ANPK does not modulate PR- and GR-dependent transcription. (B) CV-1 cells were transfected with 5 μg of pARE 2 -E1b-CAT reporter containing two copies of the GRE/PRE/ARE motif of the rat tyrosine aminotransferase gene upstream of the adenovirus E1b TATA sequence along with 0.5 μg of pSG5-hGR, 5 μg of empty expression vector (pFLAG-CMV-2) (open bar) or pFLAG-ANPK(159–1191) (solid bar), and 2 μg of pCMVβ in the presence or absence of 100 nM dexamethasone (D). (C) CV-1 cells were transfected as in panel B, but using 0.5 μg of pSG5-hPR1 instead of pSG5-hGR in the presence or absence of 100 nM progesterone (P). CAT activities are normalized to β-gal activity and expressed relative to those achieved with pSG5-hGR or pSG5-hPR1 in the presence of P or D, respectively (= 100), and the mean ± SE values of at least three independent experiments are shown.

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

Influence of ANPK on the function of various AR mutants. (A) Structural features of AR mutants studied. (B) Effect of ANPK on AR mutants was examined in CV-1 cells by coexpressing rAR or the deletion mutants rAR▵40–147, rAR▵641–902, and rAR▵46–408/▵641–902 (0.5 μg of each pSG5 expression vector) in the presence of empty pFLAG-CMV2 expression vector (5 μg, open bars) or with pFLAG-ANPK(159–1191) (5 μg, solid bars) and 5 μg of pARE 2 -E1b-CAT reporter in the presence of 100 nM testosterone. Cells were transiently transfected using the calcium phosphate method. β-Gal expression plasmid, pCMVβ (2 μg/10-cm plate), was used to control for transfection efficiency. CAT activities are expressed relative to that of pSG5-rAR in the presence of testosterone (= 100), and the mean ± SE values of at least three independent experiments are given.
Figure Legend Snippet: Influence of ANPK on the function of various AR mutants. (A) Structural features of AR mutants studied. (B) Effect of ANPK on AR mutants was examined in CV-1 cells by coexpressing rAR or the deletion mutants rAR▵40–147, rAR▵641–902, and rAR▵46–408/▵641–902 (0.5 μg of each pSG5 expression vector) in the presence of empty pFLAG-CMV2 expression vector (5 μg, open bars) or with pFLAG-ANPK(159–1191) (5 μg, solid bars) and 5 μg of pARE 2 -E1b-CAT reporter in the presence of 100 nM testosterone. Cells were transiently transfected using the calcium phosphate method. β-Gal expression plasmid, pCMVβ (2 μg/10-cm plate), was used to control for transfection efficiency. CAT activities are expressed relative to that of pSG5-rAR in the presence of testosterone (= 100), and the mean ± SE values of at least three independent experiments are given.

Techniques Used: Expressing, Plasmid Preparation, Transfection

3) Product Images from "Identification of a Novel RING Finger Protein as a Coregulator in Steroid Receptor-Mediated Gene Transcription"

Article Title: Identification of a Novel RING Finger Protein as a Coregulator in Steroid Receptor-Mediated Gene Transcription

Journal: Molecular and Cellular Biology

doi:

Interaction between AR and SNURF in mammalian cells. (A) The ability of rAR (residues 3 to 902) fused to the DBD of Gal4 (Gal4-AR) to interact with VP16 AD fused to SNURF residues 20 to 177 (VP16-SNURF) or to polyomavirus coat protein (VP16-CP) was examined in CV-1 cells by assaying chloramphenicol acetyltransferase (CAT) activity from the reporter plasmid pG5CAT. Cells (2.3 × 10 5 ). (B) AR and SNURF are physically associated in COS-1 cells. COS-1 cells were transfected by electroporation with pFLAG-SNURF or pFLAG-SNURFΔID and pSG5-rAR as indicated. After a 30-h culture in the presence of 100 nM testosterone, whole-cell extracts were prepared and subjected to immunoprecipitation (IP) with mouse monoclonal anti-FLAG antibody. Immunoprecipitated proteins were analyzed by immunoblotting with a rabbit anti-AR antibody. Lanes 1 to 4 (input) represent portions of the cell extracts (5%) that were subjected to immunoblotting without prior immunoprecipitation.
Figure Legend Snippet: Interaction between AR and SNURF in mammalian cells. (A) The ability of rAR (residues 3 to 902) fused to the DBD of Gal4 (Gal4-AR) to interact with VP16 AD fused to SNURF residues 20 to 177 (VP16-SNURF) or to polyomavirus coat protein (VP16-CP) was examined in CV-1 cells by assaying chloramphenicol acetyltransferase (CAT) activity from the reporter plasmid pG5CAT. Cells (2.3 × 10 5 ). (B) AR and SNURF are physically associated in COS-1 cells. COS-1 cells were transfected by electroporation with pFLAG-SNURF or pFLAG-SNURFΔID and pSG5-rAR as indicated. After a 30-h culture in the presence of 100 nM testosterone, whole-cell extracts were prepared and subjected to immunoprecipitation (IP) with mouse monoclonal anti-FLAG antibody. Immunoprecipitated proteins were analyzed by immunoblotting with a rabbit anti-AR antibody. Lanes 1 to 4 (input) represent portions of the cell extracts (5%) that were subjected to immunoblotting without prior immunoprecipitation.

Techniques Used: Activity Assay, Plasmid Preparation, Transfection, Electroporation, Immunoprecipitation

4) Product Images from "Identification of a Novel RING Finger Protein as a Coregulator in Steroid Receptor-Mediated Gene Transcription"

Article Title: Identification of a Novel RING Finger Protein as a Coregulator in Steroid Receptor-Mediated Gene Transcription

Journal: Molecular and Cellular Biology

doi:

RING finger mutated SNURF is capable of enhancing AR-dependent transactivation but not basal transcription. (A) Influence of SNURF and SNURF(C→S) on transcription from the AR-dependent probasin promoter. CV-1 cells were transfected with 5 μg of pPB(−285/+32)-LUC reporter, 1 μg of pSG5-rAR, 2 μg of pCMVβ, and 5 μg of expression vectors of SNURF, SNURF(C→S), or SNURFΔID/(C→S) or the empty expression vector (pcDNA-3.1+) in the presence or absence of 100 nM testosterone (T) as indicated. (B) Transcription from a minimal AR-dependent promoter. Experimental conditions were as described for panel A, except that 5 μg of pARE 2 -TATA-LUC was used as the reporter. (C) Effect of SNURF and SNURF(C→S) on transcription from a minimal TATA-LUC promoter. Experimental conditions were as described for panel A, except that 5 μg of pTATA-LUC in the absence of pSG5-rAR was used. (D) Influence of SNURF and SNURF(C→S) on transcription from a simple Sp1-AP1-TATA promoter. CV-1 cells were transfected as described above but in the absence of pSG5-rAR, and 5 μg of pSp1-AP1-TATA-LUC containing a Sp1 and an AP1 binding site upstream of TATA sequence was used as the reporter. The amounts of pcDNA-SNURF and pcDNA-SNURF(C→S) are given in micrograms. Luciferase (LUC) activities were normalized for transfection efficiency by using the β-galactosidase activity.
Figure Legend Snippet: RING finger mutated SNURF is capable of enhancing AR-dependent transactivation but not basal transcription. (A) Influence of SNURF and SNURF(C→S) on transcription from the AR-dependent probasin promoter. CV-1 cells were transfected with 5 μg of pPB(−285/+32)-LUC reporter, 1 μg of pSG5-rAR, 2 μg of pCMVβ, and 5 μg of expression vectors of SNURF, SNURF(C→S), or SNURFΔID/(C→S) or the empty expression vector (pcDNA-3.1+) in the presence or absence of 100 nM testosterone (T) as indicated. (B) Transcription from a minimal AR-dependent promoter. Experimental conditions were as described for panel A, except that 5 μg of pARE 2 -TATA-LUC was used as the reporter. (C) Effect of SNURF and SNURF(C→S) on transcription from a minimal TATA-LUC promoter. Experimental conditions were as described for panel A, except that 5 μg of pTATA-LUC in the absence of pSG5-rAR was used. (D) Influence of SNURF and SNURF(C→S) on transcription from a simple Sp1-AP1-TATA promoter. CV-1 cells were transfected as described above but in the absence of pSG5-rAR, and 5 μg of pSp1-AP1-TATA-LUC containing a Sp1 and an AP1 binding site upstream of TATA sequence was used as the reporter. The amounts of pcDNA-SNURF and pcDNA-SNURF(C→S) are given in micrograms. Luciferase (LUC) activities were normalized for transfection efficiency by using the β-galactosidase activity.

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

Influence of SNURF overexpression on AR-dependent transactivation. (A) SNURF enhances AR-dependent and basal transcription from the rat probasin promoter. COS-1 cells were transfected by the calcium phosphate method with 5 μg of pPB(−285/+32)-LUC reporter plasmid along with 1 μg of pSG5-rAR or empty pSG5 and 5 μg of SNURF expression vector (pcDNA-SNURF) or empty expression vector (pcDNA-3.1+) in the presence 25 nM testosterone (T) as depicted. β-Galactosidase expression plasmid, pCMVβ (2 μg), was used as a control for transfection efficiency. (B) CV-1 cells were transfected with 1 μg of pSG5-rAR, 5 μg of pARE 2 -TATA-LUC reporter, 2 μg of pCMVβ, and 5 μg of pcDNA-SNURF or empty expression vector in the presence or absence of testosterone (T) as depicted. (C) The experimental conditions were as in panel B, except that 5 μg of pTATA-LUC (devoid of AREs) was used as a reporter. Reporter gene activities are expressed relative to that achieved with pSG5-rAR in the presence of testosterone (100 in panels A and B; 1 in panel C), and the mean ± standard error values of at least three independent experiments are given. (D and E) SNURF activates PR- and GR-dependent transcription. (D) Effect of SNURF on the transcriptional activity of PR. CV-1 cells were transfected with 5 μg of pARE 2 -tk-LUC reporter containing two copies of the GRE-PRE-ARE element of the rat TAT gene upstream of the thymidine kinase promoter along with 1 μg of pSG5-hPR1, 5 μg of pcDNA-SNURF or empty expression vector pcDNA-3.1+, and 2 μg of pCMVβ in the presence or absence of 100 nM progesterone (P). (E) CV-1 cells were transfected as for panel D but with 1 μg of pSG5-hGR instead of pSG5-hPR1 in the presence or absence of 100 nM dexamethasone (D). Luciferase (LUC) activities are expressed relative to those achieved with pSG5-hPR1 and pSG5-hGR in the presence of progesterone and dexamethasone, respectively (those values being equal to 100), and the mean ± standard error values of at least three independent experiments are shown. (F) Effect of SNURF overexpression on Sp1 activity. CV-1 cells were transiently transfected with 5 μg of pTATA-LUC, pSp1-TATA-LUC, or pSp1 2 -TATA-LUC reporters along with 5 μg of SNURF expression vector (pFLAG-SNURF) or empty expression vector (pFLAG-CMV-2) and also 2 μg of pCMVβ. Transcriptional activities are expressed as relative luciferase (LUC) activity normalized by using the β-galactosidase activity.
Figure Legend Snippet: Influence of SNURF overexpression on AR-dependent transactivation. (A) SNURF enhances AR-dependent and basal transcription from the rat probasin promoter. COS-1 cells were transfected by the calcium phosphate method with 5 μg of pPB(−285/+32)-LUC reporter plasmid along with 1 μg of pSG5-rAR or empty pSG5 and 5 μg of SNURF expression vector (pcDNA-SNURF) or empty expression vector (pcDNA-3.1+) in the presence 25 nM testosterone (T) as depicted. β-Galactosidase expression plasmid, pCMVβ (2 μg), was used as a control for transfection efficiency. (B) CV-1 cells were transfected with 1 μg of pSG5-rAR, 5 μg of pARE 2 -TATA-LUC reporter, 2 μg of pCMVβ, and 5 μg of pcDNA-SNURF or empty expression vector in the presence or absence of testosterone (T) as depicted. (C) The experimental conditions were as in panel B, except that 5 μg of pTATA-LUC (devoid of AREs) was used as a reporter. Reporter gene activities are expressed relative to that achieved with pSG5-rAR in the presence of testosterone (100 in panels A and B; 1 in panel C), and the mean ± standard error values of at least three independent experiments are given. (D and E) SNURF activates PR- and GR-dependent transcription. (D) Effect of SNURF on the transcriptional activity of PR. CV-1 cells were transfected with 5 μg of pARE 2 -tk-LUC reporter containing two copies of the GRE-PRE-ARE element of the rat TAT gene upstream of the thymidine kinase promoter along with 1 μg of pSG5-hPR1, 5 μg of pcDNA-SNURF or empty expression vector pcDNA-3.1+, and 2 μg of pCMVβ in the presence or absence of 100 nM progesterone (P). (E) CV-1 cells were transfected as for panel D but with 1 μg of pSG5-hGR instead of pSG5-hPR1 in the presence or absence of 100 nM dexamethasone (D). Luciferase (LUC) activities are expressed relative to those achieved with pSG5-hPR1 and pSG5-hGR in the presence of progesterone and dexamethasone, respectively (those values being equal to 100), and the mean ± standard error values of at least three independent experiments are shown. (F) Effect of SNURF overexpression on Sp1 activity. CV-1 cells were transiently transfected with 5 μg of pTATA-LUC, pSp1-TATA-LUC, or pSp1 2 -TATA-LUC reporters along with 5 μg of SNURF expression vector (pFLAG-SNURF) or empty expression vector (pFLAG-CMV-2) and also 2 μg of pCMVβ. Transcriptional activities are expressed as relative luciferase (LUC) activity normalized by using the β-galactosidase activity.

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

5) Product Images from "Expression of Human Herpesvirus 6B rep within Infected Cells and Binding of Its Gene Product to the TATA-Binding Protein In Vitro and In Vivo"

Article Title: Expression of Human Herpesvirus 6B rep within Infected Cells and Binding of Its Gene Product to the TATA-Binding Protein In Vitro and In Vivo

Journal: Journal of Virology

doi:

REP binds hTBP in vivo as determined by GAL4 two-hybrid system. All of the negative controls resulted in relatively low signals (pG5 [reporter plasmid pG5 E1b Luc] + pM [GAL4 DNA binding domain-based expression plasmid] + pVP16 [HSV VP16 transactivation domain-based expression plasmid], pG5 + pMhTBP + pVP16, pG5 + pM + pVP16rep or pG5). In contrast, both the positive control (pG5 + pM53 [p53]; + pVP16-T [large T]) and pG5 + PMhTBP + pVP16rep gave significantly higher signals. Furthermore, pVP16 RepΔEco+pMhTBP showed higher luciferase activity than pVP16 Rep+pMhTBP. On the other hand, pVP RepΔMlu showed much higher activity than either pVP16 Rep or pVP16 RepΔEco. Thus, these data are consistent with a significant REP-TBP interaction in vivo. Data (means + standard deviations [error bars] of the relative luciferase activity) were calculated from triplicate cultures and are representative of three independent experiments.
Figure Legend Snippet: REP binds hTBP in vivo as determined by GAL4 two-hybrid system. All of the negative controls resulted in relatively low signals (pG5 [reporter plasmid pG5 E1b Luc] + pM [GAL4 DNA binding domain-based expression plasmid] + pVP16 [HSV VP16 transactivation domain-based expression plasmid], pG5 + pMhTBP + pVP16, pG5 + pM + pVP16rep or pG5). In contrast, both the positive control (pG5 + pM53 [p53]; + pVP16-T [large T]) and pG5 + PMhTBP + pVP16rep gave significantly higher signals. Furthermore, pVP16 RepΔEco+pMhTBP showed higher luciferase activity than pVP16 Rep+pMhTBP. On the other hand, pVP RepΔMlu showed much higher activity than either pVP16 Rep or pVP16 RepΔEco. Thus, these data are consistent with a significant REP-TBP interaction in vivo. Data (means + standard deviations [error bars] of the relative luciferase activity) were calculated from triplicate cultures and are representative of three independent experiments.

Techniques Used: In Vivo, Plasmid Preparation, Binding Assay, Expressing, Positive Control, Luciferase, Activity Assay

6) Product Images from "Activation of Androgen Receptor Function by a Novel Nuclear Protein Kinase"

Article Title: Activation of Androgen Receptor Function by a Novel Nuclear Protein Kinase

Journal: Molecular Biology of the Cell

doi:

Interaction between AR and ANPK in yeast and mammalian cells and in vitro. (A) Plasmids expressing LexA or LexA fused to full-length AR (LexA-AR), AR ZFR including part of the hinge region (LexA-ZFR), AR ZFR without hinge region sequences (LexA-ZFR-s) or AR hinge-LBD fragment (LexA-HLBD) were introduced into L40 yeast cells together with expression plasmids for VP16 AD and VP16 AD fused to ANPK(766–920) (VP16-ANPK-ID). Transformants were grown in the presence (+) or absence (–) of 50 nM testosterone (Test). β-Gal activity in extracts from liquid yeast cultures are shown. Each bar depicts the average of three independent yeast transformants. Immunoblot and whole-cell ligand-binding assays indicated that the LexA-AR fusion proteins examined were expressed to comparable levels (our unpublished data). (B) Interaction of AR with ANPK in CV-1 cells. The ability of rAR and the DBD of Gal4 (Gal4-AR) as a fusion protein to interact with the residues 159–920 of ANPK fused to VP16 AD (VP16-ANPK) or with polyoma virus coat protein fused to VP16 AD (VP16-CP) was examined in CV-1 cells using the reporter plasmid pG5CAT. Cells (2.3 × 10 5 cells/35-mm dish) were transfected with 1.5 μg of each chimeric expression vector and 3 μg of pG5CAT reporter using DOTAP transfection reagent. Eighteen hours after transfection, the medium was changed to one containing charcoal-stripped 2% (vol/vol) FBS in the presence (+) or absence (–) of 100 nM testosterone (T), and the cells were incubated for an additional 30 h. Transcriptional activation is expressed as the relative CAT activity corrected for protein concentration. Mean ± SE values for at least three separate experiments are shown. (C) Specific interaction of ANPK and AR ZFR in vitro. 35 S-Labeled full-length ANPK was synthesized by translation in vitro using reticulocyte lysate and incubated with GST alone (lane 2) or GST-AR ZFR (lane 3) adsorbed onto Glutathione Sepharose, after which the matrix was washed and bound proteins were analyzed as described in MATERIALS AND METHODS. Lane 1 represents 15% of the amount of labeled ANPK incubated with the matrix.
Figure Legend Snippet: Interaction between AR and ANPK in yeast and mammalian cells and in vitro. (A) Plasmids expressing LexA or LexA fused to full-length AR (LexA-AR), AR ZFR including part of the hinge region (LexA-ZFR), AR ZFR without hinge region sequences (LexA-ZFR-s) or AR hinge-LBD fragment (LexA-HLBD) were introduced into L40 yeast cells together with expression plasmids for VP16 AD and VP16 AD fused to ANPK(766–920) (VP16-ANPK-ID). Transformants were grown in the presence (+) or absence (–) of 50 nM testosterone (Test). β-Gal activity in extracts from liquid yeast cultures are shown. Each bar depicts the average of three independent yeast transformants. Immunoblot and whole-cell ligand-binding assays indicated that the LexA-AR fusion proteins examined were expressed to comparable levels (our unpublished data). (B) Interaction of AR with ANPK in CV-1 cells. The ability of rAR and the DBD of Gal4 (Gal4-AR) as a fusion protein to interact with the residues 159–920 of ANPK fused to VP16 AD (VP16-ANPK) or with polyoma virus coat protein fused to VP16 AD (VP16-CP) was examined in CV-1 cells using the reporter plasmid pG5CAT. Cells (2.3 × 10 5 cells/35-mm dish) were transfected with 1.5 μg of each chimeric expression vector and 3 μg of pG5CAT reporter using DOTAP transfection reagent. Eighteen hours after transfection, the medium was changed to one containing charcoal-stripped 2% (vol/vol) FBS in the presence (+) or absence (–) of 100 nM testosterone (T), and the cells were incubated for an additional 30 h. Transcriptional activation is expressed as the relative CAT activity corrected for protein concentration. Mean ± SE values for at least three separate experiments are shown. (C) Specific interaction of ANPK and AR ZFR in vitro. 35 S-Labeled full-length ANPK was synthesized by translation in vitro using reticulocyte lysate and incubated with GST alone (lane 2) or GST-AR ZFR (lane 3) adsorbed onto Glutathione Sepharose, after which the matrix was washed and bound proteins were analyzed as described in MATERIALS AND METHODS. Lane 1 represents 15% of the amount of labeled ANPK incubated with the matrix.

Techniques Used: In Vitro, Expressing, Activity Assay, Ligand Binding Assay, Plasmid Preparation, Transfection, Incubation, Activation Assay, Protein Concentration, Labeling, Synthesized

ANPK enhances androgen-induced transcriptional activation. (A) CV-1 cells were transfected using the calcium phosphate method with 5 μg of pPB(-285/+32)-LUC reporter plasmid along with 0.5 μg of pSG5-rAR and indicated amounts (μg) of pFLAG-ANPK(159–1191) or kinase-defective pFLAG-ANPK(K226R) in the presence or absence of 100 nM testosterone (T) as depicted. Total amount of DNA was kept constant by adding empty pFLAG-CMV-2 expression vector as needed. β-Gal expression plasmid, pCMVβ (2 μg/10-cm plate), was used to control for transfection efficiency. Luciferase (LUC) activities were normalized using β-gal activity. LUC activities are expressed relative to that of pSG5-rAR in the presence of testosterone (= 100), and the mean ± SE values of at least six independent experiments are given. (B and C) ANPK does not modulate PR- and GR-dependent transcription. (B) CV-1 cells were transfected with 5 μg of pARE 2 -E1b-CAT reporter containing two copies of the GRE/PRE/ARE motif of the rat tyrosine aminotransferase gene upstream of the adenovirus E1b TATA sequence along with 0.5 μg of pSG5-hGR, 5 μg of empty expression vector (pFLAG-CMV-2) (open bar) or pFLAG-ANPK(159–1191) (solid bar), and 2 μg of pCMVβ in the presence or absence of 100 nM dexamethasone (D). (C) CV-1 cells were transfected as in panel B, but using 0.5 μg of pSG5-hPR1 instead of pSG5-hGR in the presence or absence of 100 nM progesterone (P). CAT activities are normalized to β-gal activity and expressed relative to those achieved with pSG5-hGR or pSG5-hPR1 in the presence of P or D, respectively (= 100), and the mean ± SE values of at least three independent experiments are shown.
Figure Legend Snippet: ANPK enhances androgen-induced transcriptional activation. (A) CV-1 cells were transfected using the calcium phosphate method with 5 μg of pPB(-285/+32)-LUC reporter plasmid along with 0.5 μg of pSG5-rAR and indicated amounts (μg) of pFLAG-ANPK(159–1191) or kinase-defective pFLAG-ANPK(K226R) in the presence or absence of 100 nM testosterone (T) as depicted. Total amount of DNA was kept constant by adding empty pFLAG-CMV-2 expression vector as needed. β-Gal expression plasmid, pCMVβ (2 μg/10-cm plate), was used to control for transfection efficiency. Luciferase (LUC) activities were normalized using β-gal activity. LUC activities are expressed relative to that of pSG5-rAR in the presence of testosterone (= 100), and the mean ± SE values of at least six independent experiments are given. (B and C) ANPK does not modulate PR- and GR-dependent transcription. (B) CV-1 cells were transfected with 5 μg of pARE 2 -E1b-CAT reporter containing two copies of the GRE/PRE/ARE motif of the rat tyrosine aminotransferase gene upstream of the adenovirus E1b TATA sequence along with 0.5 μg of pSG5-hGR, 5 μg of empty expression vector (pFLAG-CMV-2) (open bar) or pFLAG-ANPK(159–1191) (solid bar), and 2 μg of pCMVβ in the presence or absence of 100 nM dexamethasone (D). (C) CV-1 cells were transfected as in panel B, but using 0.5 μg of pSG5-hPR1 instead of pSG5-hGR in the presence or absence of 100 nM progesterone (P). CAT activities are normalized to β-gal activity and expressed relative to those achieved with pSG5-hGR or pSG5-hPR1 in the presence of P or D, respectively (= 100), and the mean ± SE values of at least three independent experiments are shown.

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

Influence of ANPK on the function of various AR mutants. (A) Structural features of AR mutants studied. (B) Effect of ANPK on AR mutants was examined in CV-1 cells by coexpressing rAR or the deletion mutants rAR▵40–147, rAR▵641–902, and rAR▵46–408/▵641–902 (0.5 μg of each pSG5 expression vector) in the presence of empty pFLAG-CMV2 expression vector (5 μg, open bars) or with pFLAG-ANPK(159–1191) (5 μg, solid bars) and 5 μg of pARE 2 -E1b-CAT reporter in the presence of 100 nM testosterone. Cells were transiently transfected using the calcium phosphate method. β-Gal expression plasmid, pCMVβ (2 μg/10-cm plate), was used to control for transfection efficiency. CAT activities are expressed relative to that of pSG5-rAR in the presence of testosterone (= 100), and the mean ± SE values of at least three independent experiments are given.
Figure Legend Snippet: Influence of ANPK on the function of various AR mutants. (A) Structural features of AR mutants studied. (B) Effect of ANPK on AR mutants was examined in CV-1 cells by coexpressing rAR or the deletion mutants rAR▵40–147, rAR▵641–902, and rAR▵46–408/▵641–902 (0.5 μg of each pSG5 expression vector) in the presence of empty pFLAG-CMV2 expression vector (5 μg, open bars) or with pFLAG-ANPK(159–1191) (5 μg, solid bars) and 5 μg of pARE 2 -E1b-CAT reporter in the presence of 100 nM testosterone. Cells were transiently transfected using the calcium phosphate method. β-Gal expression plasmid, pCMVβ (2 μg/10-cm plate), was used to control for transfection efficiency. CAT activities are expressed relative to that of pSG5-rAR in the presence of testosterone (= 100), and the mean ± SE values of at least three independent experiments are given.

Techniques Used: Expressing, Plasmid Preparation, Transfection

7) Product Images from "Activation of Androgen Receptor Function by a Novel Nuclear Protein Kinase"

Article Title: Activation of Androgen Receptor Function by a Novel Nuclear Protein Kinase

Journal: Molecular Biology of the Cell

doi:

Interaction between AR and ANPK in yeast and mammalian cells and in vitro. (A) Plasmids expressing LexA or LexA fused to full-length AR (LexA-AR), AR ZFR including part of the hinge region (LexA-ZFR), AR ZFR without hinge region sequences (LexA-ZFR-s) or AR hinge-LBD fragment (LexA-HLBD) were introduced into L40 yeast cells together with expression plasmids for VP16 AD and VP16 AD fused to ANPK(766–920) (VP16-ANPK-ID). Transformants were grown in the presence (+) or absence (–) of 50 nM testosterone (Test). β-Gal activity in extracts from liquid yeast cultures are shown. Each bar depicts the average of three independent yeast transformants. Immunoblot and whole-cell ligand-binding assays indicated that the LexA-AR fusion proteins examined were expressed to comparable levels (our unpublished data). (B) Interaction of AR with ANPK in CV-1 cells. The ability of rAR and the DBD of Gal4 (Gal4-AR) as a fusion protein to interact with the residues 159–920 of ANPK fused to VP16 AD (VP16-ANPK) or with polyoma virus coat protein fused to VP16 AD (VP16-CP) was examined in CV-1 cells using the reporter plasmid pG5CAT. Cells (2.3 × 10 5 cells/35-mm dish) were transfected with 1.5 μg of each chimeric expression vector and 3 μg of pG5CAT reporter using DOTAP transfection reagent. Eighteen hours after transfection, the medium was changed to one containing charcoal-stripped 2% (vol/vol) FBS in the presence (+) or absence (–) of 100 nM testosterone (T), and the cells were incubated for an additional 30 h. Transcriptional activation is expressed as the relative CAT activity corrected for protein concentration. Mean ± SE values for at least three separate experiments are shown. (C) Specific interaction of ANPK and AR ZFR in vitro. 35 S-Labeled full-length ANPK was synthesized by translation in vitro using reticulocyte lysate and incubated with GST alone (lane 2) or GST-AR ZFR (lane 3) adsorbed onto Glutathione Sepharose, after which the matrix was washed and bound proteins were analyzed as described in MATERIALS AND METHODS. Lane 1 represents 15% of the amount of labeled ANPK incubated with the matrix.
Figure Legend Snippet: Interaction between AR and ANPK in yeast and mammalian cells and in vitro. (A) Plasmids expressing LexA or LexA fused to full-length AR (LexA-AR), AR ZFR including part of the hinge region (LexA-ZFR), AR ZFR without hinge region sequences (LexA-ZFR-s) or AR hinge-LBD fragment (LexA-HLBD) were introduced into L40 yeast cells together with expression plasmids for VP16 AD and VP16 AD fused to ANPK(766–920) (VP16-ANPK-ID). Transformants were grown in the presence (+) or absence (–) of 50 nM testosterone (Test). β-Gal activity in extracts from liquid yeast cultures are shown. Each bar depicts the average of three independent yeast transformants. Immunoblot and whole-cell ligand-binding assays indicated that the LexA-AR fusion proteins examined were expressed to comparable levels (our unpublished data). (B) Interaction of AR with ANPK in CV-1 cells. The ability of rAR and the DBD of Gal4 (Gal4-AR) as a fusion protein to interact with the residues 159–920 of ANPK fused to VP16 AD (VP16-ANPK) or with polyoma virus coat protein fused to VP16 AD (VP16-CP) was examined in CV-1 cells using the reporter plasmid pG5CAT. Cells (2.3 × 10 5 cells/35-mm dish) were transfected with 1.5 μg of each chimeric expression vector and 3 μg of pG5CAT reporter using DOTAP transfection reagent. Eighteen hours after transfection, the medium was changed to one containing charcoal-stripped 2% (vol/vol) FBS in the presence (+) or absence (–) of 100 nM testosterone (T), and the cells were incubated for an additional 30 h. Transcriptional activation is expressed as the relative CAT activity corrected for protein concentration. Mean ± SE values for at least three separate experiments are shown. (C) Specific interaction of ANPK and AR ZFR in vitro. 35 S-Labeled full-length ANPK was synthesized by translation in vitro using reticulocyte lysate and incubated with GST alone (lane 2) or GST-AR ZFR (lane 3) adsorbed onto Glutathione Sepharose, after which the matrix was washed and bound proteins were analyzed as described in MATERIALS AND METHODS. Lane 1 represents 15% of the amount of labeled ANPK incubated with the matrix.

Techniques Used: In Vitro, Expressing, Activity Assay, Ligand Binding Assay, Plasmid Preparation, Transfection, Incubation, Activation Assay, Protein Concentration, Labeling, Synthesized

ANPK enhances androgen-induced transcriptional activation. (A) CV-1 cells were transfected using the calcium phosphate method with 5 μg of pPB(-285/+32)-LUC reporter plasmid along with 0.5 μg of pSG5-rAR and indicated amounts (μg) of pFLAG-ANPK(159–1191) or kinase-defective pFLAG-ANPK(K226R) in the presence or absence of 100 nM testosterone (T) as depicted. Total amount of DNA was kept constant by adding empty pFLAG-CMV-2 expression vector as needed. β-Gal expression plasmid, pCMVβ (2 μg/10-cm plate), was used to control for transfection efficiency. Luciferase (LUC) activities were normalized using β-gal activity. LUC activities are expressed relative to that of pSG5-rAR in the presence of testosterone (= 100), and the mean ± SE values of at least six independent experiments are given. (B and C) ANPK does not modulate PR- and GR-dependent transcription. (B) CV-1 cells were transfected with 5 μg of pARE 2 -E1b-CAT reporter containing two copies of the GRE/PRE/ARE motif of the rat tyrosine aminotransferase gene upstream of the adenovirus E1b TATA sequence along with 0.5 μg of pSG5-hGR, 5 μg of empty expression vector (pFLAG-CMV-2) (open bar) or pFLAG-ANPK(159–1191) (solid bar), and 2 μg of pCMVβ in the presence or absence of 100 nM dexamethasone (D). (C) CV-1 cells were transfected as in panel B, but using 0.5 μg of pSG5-hPR1 instead of pSG5-hGR in the presence or absence of 100 nM progesterone (P). CAT activities are normalized to β-gal activity and expressed relative to those achieved with pSG5-hGR or pSG5-hPR1 in the presence of P or D, respectively (= 100), and the mean ± SE values of at least three independent experiments are shown.
Figure Legend Snippet: ANPK enhances androgen-induced transcriptional activation. (A) CV-1 cells were transfected using the calcium phosphate method with 5 μg of pPB(-285/+32)-LUC reporter plasmid along with 0.5 μg of pSG5-rAR and indicated amounts (μg) of pFLAG-ANPK(159–1191) or kinase-defective pFLAG-ANPK(K226R) in the presence or absence of 100 nM testosterone (T) as depicted. Total amount of DNA was kept constant by adding empty pFLAG-CMV-2 expression vector as needed. β-Gal expression plasmid, pCMVβ (2 μg/10-cm plate), was used to control for transfection efficiency. Luciferase (LUC) activities were normalized using β-gal activity. LUC activities are expressed relative to that of pSG5-rAR in the presence of testosterone (= 100), and the mean ± SE values of at least six independent experiments are given. (B and C) ANPK does not modulate PR- and GR-dependent transcription. (B) CV-1 cells were transfected with 5 μg of pARE 2 -E1b-CAT reporter containing two copies of the GRE/PRE/ARE motif of the rat tyrosine aminotransferase gene upstream of the adenovirus E1b TATA sequence along with 0.5 μg of pSG5-hGR, 5 μg of empty expression vector (pFLAG-CMV-2) (open bar) or pFLAG-ANPK(159–1191) (solid bar), and 2 μg of pCMVβ in the presence or absence of 100 nM dexamethasone (D). (C) CV-1 cells were transfected as in panel B, but using 0.5 μg of pSG5-hPR1 instead of pSG5-hGR in the presence or absence of 100 nM progesterone (P). CAT activities are normalized to β-gal activity and expressed relative to those achieved with pSG5-hGR or pSG5-hPR1 in the presence of P or D, respectively (= 100), and the mean ± SE values of at least three independent experiments are shown.

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

Influence of ANPK on the function of various AR mutants. (A) Structural features of AR mutants studied. (B) Effect of ANPK on AR mutants was examined in CV-1 cells by coexpressing rAR or the deletion mutants rAR▵40–147, rAR▵641–902, and rAR▵46–408/▵641–902 (0.5 μg of each pSG5 expression vector) in the presence of empty pFLAG-CMV2 expression vector (5 μg, open bars) or with pFLAG-ANPK(159–1191) (5 μg, solid bars) and 5 μg of pARE 2 -E1b-CAT reporter in the presence of 100 nM testosterone. Cells were transiently transfected using the calcium phosphate method. β-Gal expression plasmid, pCMVβ (2 μg/10-cm plate), was used to control for transfection efficiency. CAT activities are expressed relative to that of pSG5-rAR in the presence of testosterone (= 100), and the mean ± SE values of at least three independent experiments are given.
Figure Legend Snippet: Influence of ANPK on the function of various AR mutants. (A) Structural features of AR mutants studied. (B) Effect of ANPK on AR mutants was examined in CV-1 cells by coexpressing rAR or the deletion mutants rAR▵40–147, rAR▵641–902, and rAR▵46–408/▵641–902 (0.5 μg of each pSG5 expression vector) in the presence of empty pFLAG-CMV2 expression vector (5 μg, open bars) or with pFLAG-ANPK(159–1191) (5 μg, solid bars) and 5 μg of pARE 2 -E1b-CAT reporter in the presence of 100 nM testosterone. Cells were transiently transfected using the calcium phosphate method. β-Gal expression plasmid, pCMVβ (2 μg/10-cm plate), was used to control for transfection efficiency. CAT activities are expressed relative to that of pSG5-rAR in the presence of testosterone (= 100), and the mean ± SE values of at least three independent experiments are given.

Techniques Used: Expressing, Plasmid Preparation, Transfection

8) Product Images from "Activation of Androgen Receptor Function by a Novel Nuclear Protein Kinase"

Article Title: Activation of Androgen Receptor Function by a Novel Nuclear Protein Kinase

Journal: Molecular Biology of the Cell

doi:

Interaction between AR and ANPK in yeast and mammalian cells and in vitro. (A) Plasmids expressing LexA or LexA fused to full-length AR (LexA-AR), AR ZFR including part of the hinge region (LexA-ZFR), AR ZFR without hinge region sequences (LexA-ZFR-s) or AR hinge-LBD fragment (LexA-HLBD) were introduced into L40 yeast cells together with expression plasmids for VP16 AD and VP16 AD fused to ANPK(766–920) (VP16-ANPK-ID). Transformants were grown in the presence (+) or absence (–) of 50 nM testosterone (Test). β-Gal activity in extracts from liquid yeast cultures are shown. Each bar depicts the average of three independent yeast transformants. Immunoblot and whole-cell ligand-binding assays indicated that the LexA-AR fusion proteins examined were expressed to comparable levels (our unpublished data). (B) Interaction of AR with ANPK in CV-1 cells. The ability of rAR and the DBD of Gal4 (Gal4-AR) as a fusion protein to interact with the residues 159–920 of ANPK fused to VP16 AD (VP16-ANPK) or with polyoma virus coat protein fused to VP16 AD (VP16-CP) was examined in CV-1 cells using the reporter plasmid pG5CAT. Cells (2.3 × 10 5 cells/35-mm dish) were transfected with 1.5 μg of each chimeric expression vector and 3 μg of pG5CAT reporter using DOTAP transfection reagent. Eighteen hours after transfection, the medium was changed to one containing charcoal-stripped 2% (vol/vol) FBS in the presence (+) or absence (–) of 100 nM testosterone (T), and the cells were incubated for an additional 30 h. Transcriptional activation is expressed as the relative CAT activity corrected for protein concentration. Mean ± SE values for at least three separate experiments are shown. (C) Specific interaction of ANPK and AR ZFR in vitro. 35 S-Labeled full-length ANPK was synthesized by translation in vitro using reticulocyte lysate and incubated with GST alone (lane 2) or GST-AR ZFR (lane 3) adsorbed onto Glutathione Sepharose, after which the matrix was washed and bound proteins were analyzed as described in MATERIALS AND METHODS. Lane 1 represents 15% of the amount of labeled ANPK incubated with the matrix.
Figure Legend Snippet: Interaction between AR and ANPK in yeast and mammalian cells and in vitro. (A) Plasmids expressing LexA or LexA fused to full-length AR (LexA-AR), AR ZFR including part of the hinge region (LexA-ZFR), AR ZFR without hinge region sequences (LexA-ZFR-s) or AR hinge-LBD fragment (LexA-HLBD) were introduced into L40 yeast cells together with expression plasmids for VP16 AD and VP16 AD fused to ANPK(766–920) (VP16-ANPK-ID). Transformants were grown in the presence (+) or absence (–) of 50 nM testosterone (Test). β-Gal activity in extracts from liquid yeast cultures are shown. Each bar depicts the average of three independent yeast transformants. Immunoblot and whole-cell ligand-binding assays indicated that the LexA-AR fusion proteins examined were expressed to comparable levels (our unpublished data). (B) Interaction of AR with ANPK in CV-1 cells. The ability of rAR and the DBD of Gal4 (Gal4-AR) as a fusion protein to interact with the residues 159–920 of ANPK fused to VP16 AD (VP16-ANPK) or with polyoma virus coat protein fused to VP16 AD (VP16-CP) was examined in CV-1 cells using the reporter plasmid pG5CAT. Cells (2.3 × 10 5 cells/35-mm dish) were transfected with 1.5 μg of each chimeric expression vector and 3 μg of pG5CAT reporter using DOTAP transfection reagent. Eighteen hours after transfection, the medium was changed to one containing charcoal-stripped 2% (vol/vol) FBS in the presence (+) or absence (–) of 100 nM testosterone (T), and the cells were incubated for an additional 30 h. Transcriptional activation is expressed as the relative CAT activity corrected for protein concentration. Mean ± SE values for at least three separate experiments are shown. (C) Specific interaction of ANPK and AR ZFR in vitro. 35 S-Labeled full-length ANPK was synthesized by translation in vitro using reticulocyte lysate and incubated with GST alone (lane 2) or GST-AR ZFR (lane 3) adsorbed onto Glutathione Sepharose, after which the matrix was washed and bound proteins were analyzed as described in MATERIALS AND METHODS. Lane 1 represents 15% of the amount of labeled ANPK incubated with the matrix.

Techniques Used: In Vitro, Expressing, Activity Assay, Ligand Binding Assay, Plasmid Preparation, Transfection, Incubation, Activation Assay, Protein Concentration, Labeling, Synthesized

9) Product Images from "Identification of a Novel RING Finger Protein as a Coregulator in Steroid Receptor-Mediated Gene Transcription"

Article Title: Identification of a Novel RING Finger Protein as a Coregulator in Steroid Receptor-Mediated Gene Transcription

Journal: Molecular and Cellular Biology

doi:

Interaction between AR and SNURF in mammalian cells. (A) The ability of rAR (residues 3 to 902) fused to the DBD of Gal4 (Gal4-AR) to interact with VP16 AD fused to SNURF residues 20 to 177 (VP16-SNURF) or to polyomavirus coat protein (VP16-CP) was examined in CV-1 cells by assaying chloramphenicol acetyltransferase (CAT) activity from the reporter plasmid pG5CAT. Cells (2.3 × 10 5 ). (B) AR and SNURF are physically associated in COS-1 cells. COS-1 cells were transfected by electroporation with pFLAG-SNURF or pFLAG-SNURFΔID and pSG5-rAR as indicated. After a 30-h culture in the presence of 100 nM testosterone, whole-cell extracts were prepared and subjected to immunoprecipitation (IP) with mouse monoclonal anti-FLAG antibody. Immunoprecipitated proteins were analyzed by immunoblotting with a rabbit anti-AR antibody. Lanes 1 to 4 (input) represent portions of the cell extracts (5%) that were subjected to immunoblotting without prior immunoprecipitation.
Figure Legend Snippet: Interaction between AR and SNURF in mammalian cells. (A) The ability of rAR (residues 3 to 902) fused to the DBD of Gal4 (Gal4-AR) to interact with VP16 AD fused to SNURF residues 20 to 177 (VP16-SNURF) or to polyomavirus coat protein (VP16-CP) was examined in CV-1 cells by assaying chloramphenicol acetyltransferase (CAT) activity from the reporter plasmid pG5CAT. Cells (2.3 × 10 5 ). (B) AR and SNURF are physically associated in COS-1 cells. COS-1 cells were transfected by electroporation with pFLAG-SNURF or pFLAG-SNURFΔID and pSG5-rAR as indicated. After a 30-h culture in the presence of 100 nM testosterone, whole-cell extracts were prepared and subjected to immunoprecipitation (IP) with mouse monoclonal anti-FLAG antibody. Immunoprecipitated proteins were analyzed by immunoblotting with a rabbit anti-AR antibody. Lanes 1 to 4 (input) represent portions of the cell extracts (5%) that were subjected to immunoblotting without prior immunoprecipitation.

Techniques Used: Activity Assay, Plasmid Preparation, Transfection, Electroporation, Immunoprecipitation

Related Articles

Clone Assay:

Article Title: Homotypic Interaction of Bunyamwera Virus Nucleocapsid Protein
Article Snippet: .. The N ORF was cloned into the BD-containing plasmid pSG424 (41) to give pSGN, and two fusions of N with AD-containing plasmids were also constructed, one using pVP16 (Clontech) to give pVPBUNN (N-terminal AD fusion tag) and another using pVP16AASV19N (41) to give pAASN (C-terminal AD fusion tag). .. HeLa cells were transfected with 1 μg each of the N-expressing plasmids together with the reporter plasmid pG5 CAT (which contains five GAL4 binding sites upstream of the adenovirus E1b promoter [ ]) using liposomes as previously described ( ).

Transfection:

Article Title: Expression of Human Herpesvirus 6B rep within Infected Cells and Binding of Its Gene Product to the TATA-Binding Protein In Vitro and In Vivo
Article Snippet: .. A reporter plasmid, pG5 E1b Luc, which was generated by replacing the chloramphenicol acetyltransferase gene in pG5 E1b CAT (Clontech) with the firefly luciferase gene was transfected at 30 ng/well with either 0.3 μg of the pM-based or the pVP-based expression vector per well. .. These cells were harvested 24 h after transfection and lysed in lysis buffer (Picagene PGC50).

Amplification:

Article Title: Overexpression of yccL (gnsA) and ydfY (gnsB) Increases Levels of Unsaturated Fatty Acids and Suppresses both the Temperature-Sensitive fabA6 Mutation and Cold-Sensitive secG Null Mutation of Escherichia coli
Article Snippet: .. The cat gene was amplified using primers cat 5′/cat 3′ with pHSG399 (Takara Shuzo Co.) as a template. .. The upstream and downstream regions of gnsA and the cat gene were excised from the cloning vector by digesting the respective unique sites and then cloned together into pBR322, which had been cut with Eam 1105I and Cla I, to construct the ΔgnsA :: cat allele.

Fluorescence:

Article Title: Bis-Anthracycline Antibiotics Inhibit Human Immunodeficiency Virus Type 1 Transcription
Article Snippet: .. Primary HIV-1 isolates were isolated from HIV-1-infected patients after they had given informed consent, and viral stocks were grown in PHA-L- and IL-2-stimulated PBMC cultures. pLTRGFP was constructed by excising the cat gene from pU3R-III cat ( , ) by using XhoI-HindIII and inserting the EGFP open reading frame excised from pEGFP-1 (Clontech) by using the same enzymes. pLZRS-YFP-Tat was constructed by replacing EGFP with enhanced yellow fluorescence protein (EYFP; pEYFP-C1; Clontech) in the original vector pLZRS-pBMN-link-EGFP (a kind gift of M. Andersson, University of Alabama at Birmingham) by using NcoI and BsrG1 and inserting HIV-1 tat (from pBabe-puro-tat [ ]) by using BamHI and SalI. .. JNLG cells were seeded at a density of 106 cells/ml in 24-well plates and were then treated with the respective drugs.

Isolation:

Article Title: Bis-Anthracycline Antibiotics Inhibit Human Immunodeficiency Virus Type 1 Transcription
Article Snippet: .. Primary HIV-1 isolates were isolated from HIV-1-infected patients after they had given informed consent, and viral stocks were grown in PHA-L- and IL-2-stimulated PBMC cultures. pLTRGFP was constructed by excising the cat gene from pU3R-III cat ( , ) by using XhoI-HindIII and inserting the EGFP open reading frame excised from pEGFP-1 (Clontech) by using the same enzymes. pLZRS-YFP-Tat was constructed by replacing EGFP with enhanced yellow fluorescence protein (EYFP; pEYFP-C1; Clontech) in the original vector pLZRS-pBMN-link-EGFP (a kind gift of M. Andersson, University of Alabama at Birmingham) by using NcoI and BsrG1 and inserting HIV-1 tat (from pBabe-puro-tat [ ]) by using BamHI and SalI. .. JNLG cells were seeded at a density of 106 cells/ml in 24-well plates and were then treated with the respective drugs.

Construct:

Article Title: Bis-Anthracycline Antibiotics Inhibit Human Immunodeficiency Virus Type 1 Transcription
Article Snippet: .. Primary HIV-1 isolates were isolated from HIV-1-infected patients after they had given informed consent, and viral stocks were grown in PHA-L- and IL-2-stimulated PBMC cultures. pLTRGFP was constructed by excising the cat gene from pU3R-III cat ( , ) by using XhoI-HindIII and inserting the EGFP open reading frame excised from pEGFP-1 (Clontech) by using the same enzymes. pLZRS-YFP-Tat was constructed by replacing EGFP with enhanced yellow fluorescence protein (EYFP; pEYFP-C1; Clontech) in the original vector pLZRS-pBMN-link-EGFP (a kind gift of M. Andersson, University of Alabama at Birmingham) by using NcoI and BsrG1 and inserting HIV-1 tat (from pBabe-puro-tat [ ]) by using BamHI and SalI. .. JNLG cells were seeded at a density of 106 cells/ml in 24-well plates and were then treated with the respective drugs.

Article Title: Homotypic Interaction of Bunyamwera Virus Nucleocapsid Protein
Article Snippet: .. The N ORF was cloned into the BD-containing plasmid pSG424 (41) to give pSGN, and two fusions of N with AD-containing plasmids were also constructed, one using pVP16 (Clontech) to give pVPBUNN (N-terminal AD fusion tag) and another using pVP16AASV19N (41) to give pAASN (C-terminal AD fusion tag). .. HeLa cells were transfected with 1 μg each of the N-expressing plasmids together with the reporter plasmid pG5 CAT (which contains five GAL4 binding sites upstream of the adenovirus E1b promoter [ ]) using liposomes as previously described ( ).

Generated:

Article Title: Expression of Human Herpesvirus 6B rep within Infected Cells and Binding of Its Gene Product to the TATA-Binding Protein In Vitro and In Vivo
Article Snippet: .. A reporter plasmid, pG5 E1b Luc, which was generated by replacing the chloramphenicol acetyltransferase gene in pG5 E1b CAT (Clontech) with the firefly luciferase gene was transfected at 30 ng/well with either 0.3 μg of the pM-based or the pVP-based expression vector per well. .. These cells were harvested 24 h after transfection and lysed in lysis buffer (Picagene PGC50).

Luciferase:

Article Title: Expression of Human Herpesvirus 6B rep within Infected Cells and Binding of Its Gene Product to the TATA-Binding Protein In Vitro and In Vivo
Article Snippet: .. A reporter plasmid, pG5 E1b Luc, which was generated by replacing the chloramphenicol acetyltransferase gene in pG5 E1b CAT (Clontech) with the firefly luciferase gene was transfected at 30 ng/well with either 0.3 μg of the pM-based or the pVP-based expression vector per well. .. These cells were harvested 24 h after transfection and lysed in lysis buffer (Picagene PGC50).

Expressing:

Article Title: Expression of Human Herpesvirus 6B rep within Infected Cells and Binding of Its Gene Product to the TATA-Binding Protein In Vitro and In Vivo
Article Snippet: .. A reporter plasmid, pG5 E1b Luc, which was generated by replacing the chloramphenicol acetyltransferase gene in pG5 E1b CAT (Clontech) with the firefly luciferase gene was transfected at 30 ng/well with either 0.3 μg of the pM-based or the pVP-based expression vector per well. .. These cells were harvested 24 h after transfection and lysed in lysis buffer (Picagene PGC50).

Sequencing:

Article Title: Activation of Androgen Receptor Function by a Novel Nuclear Protein Kinase
Article Snippet: .. pPB(−285/+32)-LUC is a reporter that contains nucleotides (nt) −285 to +32 of the rat probasin promoter ( ) and pGRE2 -E1b-CAT (pARE2 -E1b-CAT in this report) contains two copies of rat tyrosine aminotransferase glucocorticoid/progesterone/androgen response element (GRE/PRE/ARE) inserted upstream of the adenovirus E1b TATA sequence (a gift from Dr. J. Cidlowski, NIEHS, Research Triangle Park, NC) ( ). pSG5-hPR1 and pHG0 encoding human PR1 and GR, respectively, were gifts from Dr. Pierre Chambon (INSERM, Illkirch, France). pSG5-hGR was created by inserting hGR coding sequence from pGH0 as a Bam HI fragment into the Bam HI site of pSG5 (Stratagene, La Jolla, CA). pCB6-WT18A (WT1) encoding Wilms’ tumor gene product was from Dr. Frank J. Rauscher III (Wistar Institute, Philadelphia, PA). pMOR encoding mouse ER was a gift from Dr. Malcolm G. Parker (Imperial Cancer Research Fund, London, UK). pG5-CAT contains five Gal4-binding sites in front of the adenovirus E1b minimal promoter driving the CAT gene ( CLONTECH , Palo Alto, CA). .. The β-galactosidase (β-gal) expression plasmid pCMVβ was purchased from CLONTECH .

Chloramphenicol Acetyltransferase Assay:

Article Title: Activation of Androgen Receptor Function by a Novel Nuclear Protein Kinase
Article Snippet: .. pPB(−285/+32)-LUC is a reporter that contains nucleotides (nt) −285 to +32 of the rat probasin promoter ( ) and pGRE2 -E1b-CAT (pARE2 -E1b-CAT in this report) contains two copies of rat tyrosine aminotransferase glucocorticoid/progesterone/androgen response element (GRE/PRE/ARE) inserted upstream of the adenovirus E1b TATA sequence (a gift from Dr. J. Cidlowski, NIEHS, Research Triangle Park, NC) ( ). pSG5-hPR1 and pHG0 encoding human PR1 and GR, respectively, were gifts from Dr. Pierre Chambon (INSERM, Illkirch, France). pSG5-hGR was created by inserting hGR coding sequence from pGH0 as a Bam HI fragment into the Bam HI site of pSG5 (Stratagene, La Jolla, CA). pCB6-WT18A (WT1) encoding Wilms’ tumor gene product was from Dr. Frank J. Rauscher III (Wistar Institute, Philadelphia, PA). pMOR encoding mouse ER was a gift from Dr. Malcolm G. Parker (Imperial Cancer Research Fund, London, UK). pG5-CAT contains five Gal4-binding sites in front of the adenovirus E1b minimal promoter driving the CAT gene ( CLONTECH , Palo Alto, CA). .. The β-galactosidase (β-gal) expression plasmid pCMVβ was purchased from CLONTECH .

Article Title: Expression of Human Herpesvirus 6B rep within Infected Cells and Binding of Its Gene Product to the TATA-Binding Protein In Vitro and In Vivo
Article Snippet: .. A reporter plasmid, pG5 E1b Luc, which was generated by replacing the chloramphenicol acetyltransferase gene in pG5 E1b CAT (Clontech) with the firefly luciferase gene was transfected at 30 ng/well with either 0.3 μg of the pM-based or the pVP-based expression vector per well. .. These cells were harvested 24 h after transfection and lysed in lysis buffer (Picagene PGC50).

Wilms Tumor Assay:

Article Title: Activation of Androgen Receptor Function by a Novel Nuclear Protein Kinase
Article Snippet: .. pPB(−285/+32)-LUC is a reporter that contains nucleotides (nt) −285 to +32 of the rat probasin promoter ( ) and pGRE2 -E1b-CAT (pARE2 -E1b-CAT in this report) contains two copies of rat tyrosine aminotransferase glucocorticoid/progesterone/androgen response element (GRE/PRE/ARE) inserted upstream of the adenovirus E1b TATA sequence (a gift from Dr. J. Cidlowski, NIEHS, Research Triangle Park, NC) ( ). pSG5-hPR1 and pHG0 encoding human PR1 and GR, respectively, were gifts from Dr. Pierre Chambon (INSERM, Illkirch, France). pSG5-hGR was created by inserting hGR coding sequence from pGH0 as a Bam HI fragment into the Bam HI site of pSG5 (Stratagene, La Jolla, CA). pCB6-WT18A (WT1) encoding Wilms’ tumor gene product was from Dr. Frank J. Rauscher III (Wistar Institute, Philadelphia, PA). pMOR encoding mouse ER was a gift from Dr. Malcolm G. Parker (Imperial Cancer Research Fund, London, UK). pG5-CAT contains five Gal4-binding sites in front of the adenovirus E1b minimal promoter driving the CAT gene ( CLONTECH , Palo Alto, CA). .. The β-galactosidase (β-gal) expression plasmid pCMVβ was purchased from CLONTECH .

Plasmid Preparation:

Article Title: Bis-Anthracycline Antibiotics Inhibit Human Immunodeficiency Virus Type 1 Transcription
Article Snippet: .. Primary HIV-1 isolates were isolated from HIV-1-infected patients after they had given informed consent, and viral stocks were grown in PHA-L- and IL-2-stimulated PBMC cultures. pLTRGFP was constructed by excising the cat gene from pU3R-III cat ( , ) by using XhoI-HindIII and inserting the EGFP open reading frame excised from pEGFP-1 (Clontech) by using the same enzymes. pLZRS-YFP-Tat was constructed by replacing EGFP with enhanced yellow fluorescence protein (EYFP; pEYFP-C1; Clontech) in the original vector pLZRS-pBMN-link-EGFP (a kind gift of M. Andersson, University of Alabama at Birmingham) by using NcoI and BsrG1 and inserting HIV-1 tat (from pBabe-puro-tat [ ]) by using BamHI and SalI. .. JNLG cells were seeded at a density of 106 cells/ml in 24-well plates and were then treated with the respective drugs.

Article Title: Homotypic Interaction of Bunyamwera Virus Nucleocapsid Protein
Article Snippet: .. The N ORF was cloned into the BD-containing plasmid pSG424 (41) to give pSGN, and two fusions of N with AD-containing plasmids were also constructed, one using pVP16 (Clontech) to give pVPBUNN (N-terminal AD fusion tag) and another using pVP16AASV19N (41) to give pAASN (C-terminal AD fusion tag). .. HeLa cells were transfected with 1 μg each of the N-expressing plasmids together with the reporter plasmid pG5 CAT (which contains five GAL4 binding sites upstream of the adenovirus E1b promoter [ ]) using liposomes as previously described ( ).

Article Title: Expression of Human Herpesvirus 6B rep within Infected Cells and Binding of Its Gene Product to the TATA-Binding Protein In Vitro and In Vivo
Article Snippet: .. A reporter plasmid, pG5 E1b Luc, which was generated by replacing the chloramphenicol acetyltransferase gene in pG5 E1b CAT (Clontech) with the firefly luciferase gene was transfected at 30 ng/well with either 0.3 μg of the pM-based or the pVP-based expression vector per well. .. These cells were harvested 24 h after transfection and lysed in lysis buffer (Picagene PGC50).

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    TaKaRa pg5 e1b cat
    REP binds hTBP in vivo as determined by GAL4 two-hybrid system. All of the negative controls resulted in relatively low signals <t>(pG5</t> [reporter plasmid pG5 <t>E1b</t> Luc] + pM [GAL4 DNA binding domain-based expression plasmid] + pVP16 [HSV VP16 transactivation domain-based expression plasmid], pG5 + pMhTBP + pVP16, pG5 + pM + pVP16rep or pG5). In contrast, both the positive control (pG5 + pM53 [p53]; + pVP16-T [large T]) and pG5 + PMhTBP + pVP16rep gave significantly higher signals. Furthermore, pVP16 RepΔEco+pMhTBP showed higher luciferase activity than pVP16 Rep+pMhTBP. On the other hand, pVP RepΔMlu showed much higher activity than either pVP16 Rep or pVP16 RepΔEco. Thus, these data are consistent with a significant REP-TBP interaction in vivo. Data (means + standard deviations [error bars] of the relative luciferase activity) were calculated from triplicate cultures and are representative of three independent experiments.
    Pg5 E1b Cat, supplied by TaKaRa, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pg5 e1b cat - by Bioz Stars, 2020-09
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    REP binds hTBP in vivo as determined by GAL4 two-hybrid system. All of the negative controls resulted in relatively low signals (pG5 [reporter plasmid pG5 E1b Luc] + pM [GAL4 DNA binding domain-based expression plasmid] + pVP16 [HSV VP16 transactivation domain-based expression plasmid], pG5 + pMhTBP + pVP16, pG5 + pM + pVP16rep or pG5). In contrast, both the positive control (pG5 + pM53 [p53]; + pVP16-T [large T]) and pG5 + PMhTBP + pVP16rep gave significantly higher signals. Furthermore, pVP16 RepΔEco+pMhTBP showed higher luciferase activity than pVP16 Rep+pMhTBP. On the other hand, pVP RepΔMlu showed much higher activity than either pVP16 Rep or pVP16 RepΔEco. Thus, these data are consistent with a significant REP-TBP interaction in vivo. Data (means + standard deviations [error bars] of the relative luciferase activity) were calculated from triplicate cultures and are representative of three independent experiments.

    Journal: Journal of Virology

    Article Title: Expression of Human Herpesvirus 6B rep within Infected Cells and Binding of Its Gene Product to the TATA-Binding Protein In Vitro and In Vivo

    doi:

    Figure Lengend Snippet: REP binds hTBP in vivo as determined by GAL4 two-hybrid system. All of the negative controls resulted in relatively low signals (pG5 [reporter plasmid pG5 E1b Luc] + pM [GAL4 DNA binding domain-based expression plasmid] + pVP16 [HSV VP16 transactivation domain-based expression plasmid], pG5 + pMhTBP + pVP16, pG5 + pM + pVP16rep or pG5). In contrast, both the positive control (pG5 + pM53 [p53]; + pVP16-T [large T]) and pG5 + PMhTBP + pVP16rep gave significantly higher signals. Furthermore, pVP16 RepΔEco+pMhTBP showed higher luciferase activity than pVP16 Rep+pMhTBP. On the other hand, pVP RepΔMlu showed much higher activity than either pVP16 Rep or pVP16 RepΔEco. Thus, these data are consistent with a significant REP-TBP interaction in vivo. Data (means + standard deviations [error bars] of the relative luciferase activity) were calculated from triplicate cultures and are representative of three independent experiments.

    Article Snippet: A reporter plasmid, pG5 E1b Luc, which was generated by replacing the chloramphenicol acetyltransferase gene in pG5 E1b CAT (Clontech) with the firefly luciferase gene was transfected at 30 ng/well with either 0.3 μg of the pM-based or the pVP-based expression vector per well.

    Techniques: In Vivo, Plasmid Preparation, Binding Assay, Expressing, Positive Control, Luciferase, Activity Assay