Structured Review

TaKaRa sv40 intron
Schematic diagram of recombinant HCMVs containing mutations of the MIE regulatory region. HCMV genome and its unique long (U L ) and short (U S ), internal repeat long (IR L ) and short (IR S ), terminal repeat long (TR L ) and short (TR S ), and a-sequence components are depicted. Locations of MIE regulatory region, MIE gene exons 1 to 4 (open boxes), and putative UL128 gene (open box) within U L component are shown. The MIE regulatory region of the WT is composed of proximal promoter (+1 to −64), enhancer (Enh; -65 to -550), unique region (-551 to -749), and modulator (Mod; -750 to -1140). Numerical base positions are assigned relative to start site of MIE RNAs. Recombinant HCMVs rΔ-300/-640 SVgfp , rΔ-300/-1108 SVgfp , rΔ-640/-1108 SVgfp , and r SVgfp were derived from rΔM SVgpt ). rΔ-300/-640 SVgfp , rΔ-300/-1108 SVgfp , and rΔ-640/-1108 SVgfp have deletions from -300 to -640, -300 to -1108, and -640 to -1108, respectively. An enhancerless <t>SV40</t> early promoter (−138 to +57), gfp ORF, and SV40 early <t>intron</t> and polyadenylation signal were inserted at the site of deletion. r SVgfp has the same insertion at -640 but has no deletion. rΔM SVgpt has a deletion of -640 to -1108 and insertion of the SV40 early transcription unit containing the gpt ORF.
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1) Product Images from "The Human Cytomegalovirus Major Immediate-Early Distal Enhancer Region Is Required for Efficient Viral Replication and Immediate-Early Gene Expression"

Article Title: The Human Cytomegalovirus Major Immediate-Early Distal Enhancer Region Is Required for Efficient Viral Replication and Immediate-Early Gene Expression

Journal: Journal of Virology

doi:

Schematic diagram of recombinant HCMVs containing mutations of the MIE regulatory region. HCMV genome and its unique long (U L ) and short (U S ), internal repeat long (IR L ) and short (IR S ), terminal repeat long (TR L ) and short (TR S ), and a-sequence components are depicted. Locations of MIE regulatory region, MIE gene exons 1 to 4 (open boxes), and putative UL128 gene (open box) within U L component are shown. The MIE regulatory region of the WT is composed of proximal promoter (+1 to −64), enhancer (Enh; -65 to -550), unique region (-551 to -749), and modulator (Mod; -750 to -1140). Numerical base positions are assigned relative to start site of MIE RNAs. Recombinant HCMVs rΔ-300/-640 SVgfp , rΔ-300/-1108 SVgfp , rΔ-640/-1108 SVgfp , and r SVgfp were derived from rΔM SVgpt ). rΔ-300/-640 SVgfp , rΔ-300/-1108 SVgfp , and rΔ-640/-1108 SVgfp have deletions from -300 to -640, -300 to -1108, and -640 to -1108, respectively. An enhancerless SV40 early promoter (−138 to +57), gfp ORF, and SV40 early intron and polyadenylation signal were inserted at the site of deletion. r SVgfp has the same insertion at -640 but has no deletion. rΔM SVgpt has a deletion of -640 to -1108 and insertion of the SV40 early transcription unit containing the gpt ORF.
Figure Legend Snippet: Schematic diagram of recombinant HCMVs containing mutations of the MIE regulatory region. HCMV genome and its unique long (U L ) and short (U S ), internal repeat long (IR L ) and short (IR S ), terminal repeat long (TR L ) and short (TR S ), and a-sequence components are depicted. Locations of MIE regulatory region, MIE gene exons 1 to 4 (open boxes), and putative UL128 gene (open box) within U L component are shown. The MIE regulatory region of the WT is composed of proximal promoter (+1 to −64), enhancer (Enh; -65 to -550), unique region (-551 to -749), and modulator (Mod; -750 to -1140). Numerical base positions are assigned relative to start site of MIE RNAs. Recombinant HCMVs rΔ-300/-640 SVgfp , rΔ-300/-1108 SVgfp , rΔ-640/-1108 SVgfp , and r SVgfp were derived from rΔM SVgpt ). rΔ-300/-640 SVgfp , rΔ-300/-1108 SVgfp , and rΔ-640/-1108 SVgfp have deletions from -300 to -640, -300 to -1108, and -640 to -1108, respectively. An enhancerless SV40 early promoter (−138 to +57), gfp ORF, and SV40 early intron and polyadenylation signal were inserted at the site of deletion. r SVgfp has the same insertion at -640 but has no deletion. rΔM SVgpt has a deletion of -640 to -1108 and insertion of the SV40 early transcription unit containing the gpt ORF.

Techniques Used: Recombinant, Sequencing, Derivative Assay

2) Product Images from "Adenovirus E1B 55-Kilodalton Protein Targets SMARCAL1 for Degradation during Infection and Modulates Cellular DNA Replication"

Article Title: Adenovirus E1B 55-Kilodalton Protein Targets SMARCAL1 for Degradation during Infection and Modulates Cellular DNA Replication

Journal: Journal of Virology

doi: 10.1128/JVI.00402-19

SMARCAL1 is targeted for degradation during Ad infection. A549 cells were either mock infected or infected with 10 PFU/cell of wt Ad5 or wt Ad12 and harvested at the appropriate times postinfection. (A) Ad5 cell lysates were then subjected to WB for SMARCAL1, p53, E1B-55K, E4orf6, and β-actin. (B) Ad12 cell lysates were subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of more than three independent experiments.
Figure Legend Snippet: SMARCAL1 is targeted for degradation during Ad infection. A549 cells were either mock infected or infected with 10 PFU/cell of wt Ad5 or wt Ad12 and harvested at the appropriate times postinfection. (A) Ad5 cell lysates were then subjected to WB for SMARCAL1, p53, E1B-55K, E4orf6, and β-actin. (B) Ad12 cell lysates were subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of more than three independent experiments.

Techniques Used: Infection, Western Blot

SMARCAL1 is degraded during Ad infection in an E1B-55K/E4orf6- and CRL-dependent manner. (A) A549 cells were either mock infected, infected with wt Ad5, or infected with E1B-55K ( dl 1520), E4orf3 (H5 pm 4150), or E4orf6 (H5 pm 4154) deletion virus. At 24 h and 48 h postinfection, cells were harvested and subjected to WB for SMARCAL1, p53, E1B-55K, E4orf3, E4orf6, and β-actin. (B) A549 cells were either mock infected, infected with wt Ad12, or infected with the E1B-55K ( dl 620) deletion virus. At 24 h and 48 h postinfection, cells were harvested and Western blotted for SMARCAL1, p53, E1B-55K, and β-actin. (C and D) A549 cells were either mock infected or infected with wt Ad5 or wt Ad12 in the absence or presence of 100 nM or 500 nM MLN4924. At 24 h and 48 h postinfection, cells were harvested and subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of three independent experiments.
Figure Legend Snippet: SMARCAL1 is degraded during Ad infection in an E1B-55K/E4orf6- and CRL-dependent manner. (A) A549 cells were either mock infected, infected with wt Ad5, or infected with E1B-55K ( dl 1520), E4orf3 (H5 pm 4150), or E4orf6 (H5 pm 4154) deletion virus. At 24 h and 48 h postinfection, cells were harvested and subjected to WB for SMARCAL1, p53, E1B-55K, E4orf3, E4orf6, and β-actin. (B) A549 cells were either mock infected, infected with wt Ad12, or infected with the E1B-55K ( dl 620) deletion virus. At 24 h and 48 h postinfection, cells were harvested and Western blotted for SMARCAL1, p53, E1B-55K, and β-actin. (C and D) A549 cells were either mock infected or infected with wt Ad5 or wt Ad12 in the absence or presence of 100 nM or 500 nM MLN4924. At 24 h and 48 h postinfection, cells were harvested and subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of three independent experiments.

Techniques Used: Infection, Western Blot

Generation and characterization of tetracycline-inducible Ad5 and Ad12 E1B-55K FlpIn U2OS cells. FlpIn U2OS cells were transfected with Ad5 E1B-55K and Ad12 E1B-55K pcDNA5/FRT/TO plasmids and the recombination plasmid pOG44. Cells were incubated in selection medium containing hygromycin (200 μg/ml). Individual colonies were isolated, expanded, and treated with 0.1 μg/ml doxycycline. Twenty-four h postinduction, cell lysates were harvested, separated by SDS-PAGE, and subjected to WB analysis for Ad5 and Ad12 E1B-55K. WB analyses were also performed to gauge the levels of SMARCAL1, p53, MRE11, and β-actin for Ad5 E1B-55K and Ad12 E1B-55K FlpIn U2OS cells. Data are representative of more than three independent experiments.
Figure Legend Snippet: Generation and characterization of tetracycline-inducible Ad5 and Ad12 E1B-55K FlpIn U2OS cells. FlpIn U2OS cells were transfected with Ad5 E1B-55K and Ad12 E1B-55K pcDNA5/FRT/TO plasmids and the recombination plasmid pOG44. Cells were incubated in selection medium containing hygromycin (200 μg/ml). Individual colonies were isolated, expanded, and treated with 0.1 μg/ml doxycycline. Twenty-four h postinduction, cell lysates were harvested, separated by SDS-PAGE, and subjected to WB analysis for Ad5 and Ad12 E1B-55K. WB analyses were also performed to gauge the levels of SMARCAL1, p53, MRE11, and β-actin for Ad5 E1B-55K and Ad12 E1B-55K FlpIn U2OS cells. Data are representative of more than three independent experiments.

Techniques Used: Transfection, Plasmid Preparation, Incubation, Selection, Isolation, SDS Page, Western Blot

SMARCAL1 is recruited to VRCs in an RPA-dependent and ATR- and CDK-dependent manner. (A) Microscopic images depicting the cellular localization of wt GFP-SMARCAL1, GFP-SMARCAL1-ΔP, and GFP-SMARCAL1-ΔRPA in mock-infected (i to iii), wt Ad5-infected (iv to vi), or wt Ad12-infected cells (vii to ix) 18 h postinfection. (B) Bar graph (± SEM) showing the percentage of GFP-labeled cells that are recruited to VRCs following Ad5 or Ad12 infection. n = 3 (300 cells per experiment, 900 cells in total). Only those cells that exhibited clear GFP-SMARCAL1 structures in Ad-infected cells, comparable to the known architecture of VRCs at different stages of infection, were counted as VRC positive. Data presented were subjected to analysis of variance with a two-tailed t test. For significance testing for difference in recruitment of GFP-SMARCAL1-ΔP to VRCs relative to that of the wt GFP-SMARCAL1 following Ad5 infection, P = 0.0065 (**); for difference in recruitment of GFP-SMARCAL1-ΔRPA to VRCs relative to that of wt GFP-SMARCAL1 following Ad5 infection, P = 8.8E−05 (****); for difference in recruitment of GFP-SMARCAL1-ΔP to VRCs relative to that of wt GFP-SMARCAL1 following Ad12 infection, P = 0.04 (*); for difference in recruitment of GFP-SMARCAL1-ΔRPA to VRCs relative to that of wt GFP-SMARCAL1 following Ad5 infection, P = 0.002 (***).
Figure Legend Snippet: SMARCAL1 is recruited to VRCs in an RPA-dependent and ATR- and CDK-dependent manner. (A) Microscopic images depicting the cellular localization of wt GFP-SMARCAL1, GFP-SMARCAL1-ΔP, and GFP-SMARCAL1-ΔRPA in mock-infected (i to iii), wt Ad5-infected (iv to vi), or wt Ad12-infected cells (vii to ix) 18 h postinfection. (B) Bar graph (± SEM) showing the percentage of GFP-labeled cells that are recruited to VRCs following Ad5 or Ad12 infection. n = 3 (300 cells per experiment, 900 cells in total). Only those cells that exhibited clear GFP-SMARCAL1 structures in Ad-infected cells, comparable to the known architecture of VRCs at different stages of infection, were counted as VRC positive. Data presented were subjected to analysis of variance with a two-tailed t test. For significance testing for difference in recruitment of GFP-SMARCAL1-ΔP to VRCs relative to that of the wt GFP-SMARCAL1 following Ad5 infection, P = 0.0065 (**); for difference in recruitment of GFP-SMARCAL1-ΔRPA to VRCs relative to that of wt GFP-SMARCAL1 following Ad5 infection, P = 8.8E−05 (****); for difference in recruitment of GFP-SMARCAL1-ΔP to VRCs relative to that of wt GFP-SMARCAL1 following Ad12 infection, P = 0.04 (*); for difference in recruitment of GFP-SMARCAL1-ΔRPA to VRCs relative to that of wt GFP-SMARCAL1 following Ad5 infection, P = 0.002 (***).

Techniques Used: Recombinase Polymerase Amplification, Infection, Labeling, Two Tailed Test

SMARCAL1 is phosphorylated during the early stages of Ad infection. (A) A549 cells were either mock infected, treated with MLN4924, or infected with 10 PFU/cell of wt Ad5 or wt Ad12 and harvested at 18 h postinfection. Cells were harvested in IP buffer and subjected to immunoprecipitation for SMARCAL1. Anti-SMARCAL1 immunoprecipitates collected on protein G-Sepharose were treated in the absence or presence of λ-phosphatase and then subjected to SDS-PAGE and WB for SMARCAL1. (B) SMARCAL1 was immunoprecipitated from mock-infected and wt Ad5- or wt Ad12-infected A549 cells 18 h postinfection and separated by SDS-PAGE. Protein bands excised from the gel were subjected to trypsinization and mass spectrometric analysis. Identified SMARCAL1 phosphorylated peptides from Ad-infected cells are presented. (C) S123, S129, and S173 are conserved between primates but less well conserved in lower mammals. SMARCAL1 primary sequences from a number of species were aligned using CLUSTAL Omega. Shaded areas indicate conserved residues.
Figure Legend Snippet: SMARCAL1 is phosphorylated during the early stages of Ad infection. (A) A549 cells were either mock infected, treated with MLN4924, or infected with 10 PFU/cell of wt Ad5 or wt Ad12 and harvested at 18 h postinfection. Cells were harvested in IP buffer and subjected to immunoprecipitation for SMARCAL1. Anti-SMARCAL1 immunoprecipitates collected on protein G-Sepharose were treated in the absence or presence of λ-phosphatase and then subjected to SDS-PAGE and WB for SMARCAL1. (B) SMARCAL1 was immunoprecipitated from mock-infected and wt Ad5- or wt Ad12-infected A549 cells 18 h postinfection and separated by SDS-PAGE. Protein bands excised from the gel were subjected to trypsinization and mass spectrometric analysis. Identified SMARCAL1 phosphorylated peptides from Ad-infected cells are presented. (C) S123, S129, and S173 are conserved between primates but less well conserved in lower mammals. SMARCAL1 primary sequences from a number of species were aligned using CLUSTAL Omega. Shaded areas indicate conserved residues.

Techniques Used: Infection, Immunoprecipitation, SDS Page, Western Blot

SMARCAL1 is reorganized to viral replication centers during the early stages of Ad infection. A549 cells were either mock infected (i to iii) or infected with 10 PFU/cell of wt Ad5 (iv to vi) or wt Ad12 (vii to ix). At 18 h postinfection, cells were fixed, permeabilized, and costained for SMARCAL1 and RPA2. Arrows indicate regions of RPA2/SMARCAL1 colocalization. In all instances, images were recorded using a Zeiss LSM510-Meta confocal microscope.
Figure Legend Snippet: SMARCAL1 is reorganized to viral replication centers during the early stages of Ad infection. A549 cells were either mock infected (i to iii) or infected with 10 PFU/cell of wt Ad5 (iv to vi) or wt Ad12 (vii to ix). At 18 h postinfection, cells were fixed, permeabilized, and costained for SMARCAL1 and RPA2. Arrows indicate regions of RPA2/SMARCAL1 colocalization. In all instances, images were recorded using a Zeiss LSM510-Meta confocal microscope.

Techniques Used: Infection, Microscopy

Ad E1B-55K associates with SMARCAL1 in Ad-transformed cells. (A) Ad E1B-55K and SMARCAL1 were immunoprecipitated from Ad5 HEK 293 cells (A) and Ad12 HER2 cells (B) and subjected to WB for E1B-55K and SMARCAL1. IgG, immunoglobulin control IP.
Figure Legend Snippet: Ad E1B-55K associates with SMARCAL1 in Ad-transformed cells. (A) Ad E1B-55K and SMARCAL1 were immunoprecipitated from Ad5 HEK 293 cells (A) and Ad12 HER2 cells (B) and subjected to WB for E1B-55K and SMARCAL1. IgG, immunoglobulin control IP.

Techniques Used: Transformation Assay, Immunoprecipitation, Western Blot

ATR kinase and CDKs promote SMARCAL1 degradation following Ad5 and Ad12 infection. A549 cells were either mock infected or infected with 10 PFU/cell of wt Ad5 (A and C) or wt Ad12 (B and D). Cells were then incubated in the absence or presence of ATR inhibitor (AZD6738 [ATRi], 1 μM; A and B) or ATR and CDK inhibitors (AZD6738, 1 μM and RO-3306 [CDKi], 9 μM; C and D) and harvested at the appropriate times postinfection. Cell lysates were then separated by SDS-PAGE and subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of three independent experiments.
Figure Legend Snippet: ATR kinase and CDKs promote SMARCAL1 degradation following Ad5 and Ad12 infection. A549 cells were either mock infected or infected with 10 PFU/cell of wt Ad5 (A and C) or wt Ad12 (B and D). Cells were then incubated in the absence or presence of ATR inhibitor (AZD6738 [ATRi], 1 μM; A and B) or ATR and CDK inhibitors (AZD6738, 1 μM and RO-3306 [CDKi], 9 μM; C and D) and harvested at the appropriate times postinfection. Cell lysates were then separated by SDS-PAGE and subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of three independent experiments.

Techniques Used: Infection, Incubation, SDS Page, Western Blot

3) Product Images from "Altered levels of circulating miRNAs are associated Schistosoma japonicum infection in mice"

Article Title: Altered levels of circulating miRNAs are associated Schistosoma japonicum infection in mice

Journal: Parasites & Vectors

doi: 10.1186/s13071-015-0806-5

Analysis of the levels of selected miRNAs and their target genes using qRT-PCR. a . qRT-PCR analysis of the plasma levels of selected miRNAs and their target genes; b . qRT-PCR analysis of the levels of selected miRNAs and their target genes in the livers of S. japonicum infected mice. Data illustrate representative experiments and show the mean and standard error derived from triplicate biological replicates. Pools of plasma and livers from at least four S. japonicum infected mice and four uninfected controls were used in each biological experiment. Creb1: cAMP responsive element binding protein 1. *means P ≤ 0.05 and **means P ≤ 0.01. (student’s t test analysis).
Figure Legend Snippet: Analysis of the levels of selected miRNAs and their target genes using qRT-PCR. a . qRT-PCR analysis of the plasma levels of selected miRNAs and their target genes; b . qRT-PCR analysis of the levels of selected miRNAs and their target genes in the livers of S. japonicum infected mice. Data illustrate representative experiments and show the mean and standard error derived from triplicate biological replicates. Pools of plasma and livers from at least four S. japonicum infected mice and four uninfected controls were used in each biological experiment. Creb1: cAMP responsive element binding protein 1. *means P ≤ 0.05 and **means P ≤ 0.01. (student’s t test analysis).

Techniques Used: Quantitative RT-PCR, Infection, Mouse Assay, Derivative Assay, Binding Assay

4) Product Images from "Modulation of Hepatic Granulomatous Responses by Transgene Expression of DAP12 or TREM-1-Ig Molecules"

Article Title: Modulation of Hepatic Granulomatous Responses by Transgene Expression of DAP12 or TREM-1-Ig Molecules

Journal: The American Journal of Pathology

doi:

Zymosan A-induced hepatic granuloma formation in Ad-LacZ ( A , D )-, Ad-FDAP12 ( B , E )-, and Ad-TREM-1 Ig ( C , F )-infected mice at day 3. Liver sections were subjected to H E staining ( A–C ) and immunostaining for F4/80 ( D–F ). Original magnifications, ×100.
Figure Legend Snippet: Zymosan A-induced hepatic granuloma formation in Ad-LacZ ( A , D )-, Ad-FDAP12 ( B , E )-, and Ad-TREM-1 Ig ( C , F )-infected mice at day 3. Liver sections were subjected to H E staining ( A–C ) and immunostaining for F4/80 ( D–F ). Original magnifications, ×100.

Techniques Used: Infection, Mouse Assay, Staining, Immunostaining

Zymosan A-induced hepatic granuloma formation in Ad-LacZ ( A , D )-, Ad-FDAP12 ( B , E )-, and Ad-TREM-1 Ig ( C , F )-infected mice at day 5. Liver sections were subjected to H E staining ( A–C ) and immunostaining for F4/80 ( D–F ). Original magnifications, ×100.
Figure Legend Snippet: Zymosan A-induced hepatic granuloma formation in Ad-LacZ ( A , D )-, Ad-FDAP12 ( B , E )-, and Ad-TREM-1 Ig ( C , F )-infected mice at day 5. Liver sections were subjected to H E staining ( A–C ) and immunostaining for F4/80 ( D–F ). Original magnifications, ×100.

Techniques Used: Infection, Mouse Assay, Staining, Immunostaining

Zymosan A-induced hepatic granuloma formation in Ad-LacZ ( A , D )-, Ad-FDAP12 ( B , E )-, and Ad-TREM-1 Ig ( C , F )-infected mice at day 10. Liver sections were subjected to H E staining ( A–C ) and immunostaining for F4/80 ( D–F ). Original magnifications, ×100.
Figure Legend Snippet: Zymosan A-induced hepatic granuloma formation in Ad-LacZ ( A , D )-, Ad-FDAP12 ( B , E )-, and Ad-TREM-1 Ig ( C , F )-infected mice at day 10. Liver sections were subjected to H E staining ( A–C ) and immunostaining for F4/80 ( D–F ). Original magnifications, ×100.

Techniques Used: Infection, Mouse Assay, Staining, Immunostaining

Morphological change and cell-surface phenotypic analysis of Ad-FDAP12-infected M1 cells stimulated with immobilized anti-FLAG mAb. A: Ad-LacZ-infected cells were used as a control. After pretreatment with LPS (10 μg/ml) for 15 to 17 hours, M1 cells were infected by adenoviral vector. Cells were cultured on the sonic seal slide wells coated with anti-FLAG mAb for 3 days without LPS. Cells were stained with H E and observed under the microscope. B: After pretreatment with LPS (10 μg/ml) for 15 to 17 hours, M1 cells were infected by adenoviral vectors and cultured on the dish coated with anti-FLAG mAb for 3 days without LPS. Cells were collected and stained with fluorescein isothiocyanate-conjugated anti-Mac-1 and phycoerythrin-conjugated anti-MHC class II monoclonal antibodies. Samples were analyzed by flow cytometry. Original magnification, ×200 ( A ).
Figure Legend Snippet: Morphological change and cell-surface phenotypic analysis of Ad-FDAP12-infected M1 cells stimulated with immobilized anti-FLAG mAb. A: Ad-LacZ-infected cells were used as a control. After pretreatment with LPS (10 μg/ml) for 15 to 17 hours, M1 cells were infected by adenoviral vector. Cells were cultured on the sonic seal slide wells coated with anti-FLAG mAb for 3 days without LPS. Cells were stained with H E and observed under the microscope. B: After pretreatment with LPS (10 μg/ml) for 15 to 17 hours, M1 cells were infected by adenoviral vectors and cultured on the dish coated with anti-FLAG mAb for 3 days without LPS. Cells were collected and stained with fluorescein isothiocyanate-conjugated anti-Mac-1 and phycoerythrin-conjugated anti-MHC class II monoclonal antibodies. Samples were analyzed by flow cytometry. Original magnification, ×200 ( A ).

Techniques Used: Infection, Plasmid Preparation, Cell Culture, Staining, Microscopy, Flow Cytometry, Cytometry

Higher magnification of zymosan A-induced hepatic granuloma. A–D: Ad-LacZ-treated mice; E–H: Ad-FDAP12-treated mice; and I–L B Ad-TREM-1 Ig-treated mice. PMN, Polymorphonuclear leukocyte; Mφ, macrophage; LC, lymphocyte; Epi, epithelioid cell. Liver sections were subjected to H E staining and observed at a high magnification of ×400.
Figure Legend Snippet: Higher magnification of zymosan A-induced hepatic granuloma. A–D: Ad-LacZ-treated mice; E–H: Ad-FDAP12-treated mice; and I–L B Ad-TREM-1 Ig-treated mice. PMN, Polymorphonuclear leukocyte; Mφ, macrophage; LC, lymphocyte; Epi, epithelioid cell. Liver sections were subjected to H E staining and observed at a high magnification of ×400.

Techniques Used: Mouse Assay, Staining

Enumeration of granulomas in the liver throughout time in Ad-LacZ-, Ad-FDAP12-, and Ad-TREM-1 Ig-infected mice ( n = 4). The results were analyzed using one-way analysis of variance and post hoc test. Granulomas were calculated in randomly selected 10 fields at an original magnification of ×200.
Figure Legend Snippet: Enumeration of granulomas in the liver throughout time in Ad-LacZ-, Ad-FDAP12-, and Ad-TREM-1 Ig-infected mice ( n = 4). The results were analyzed using one-way analysis of variance and post hoc test. Granulomas were calculated in randomly selected 10 fields at an original magnification of ×200.

Techniques Used: Infection, Mouse Assay

Zymosan A-induced hepatic granuloma formation in Ad-LacZ ( A , D )-, Ad-FDAP12 ( B , E )-, and Ad-TREM-1 Ig ( C , F )-infected mice at day 7. Liver sections were subjected to H E staining ( A–C ) and immunostaining for F4/80 ( D–F ). Original magnifications, ×100.
Figure Legend Snippet: Zymosan A-induced hepatic granuloma formation in Ad-LacZ ( A , D )-, Ad-FDAP12 ( B , E )-, and Ad-TREM-1 Ig ( C , F )-infected mice at day 7. Liver sections were subjected to H E staining ( A–C ) and immunostaining for F4/80 ( D–F ). Original magnifications, ×100.

Techniques Used: Infection, Mouse Assay, Staining, Immunostaining

A: Gene expression of TREM-1 and FLAG-DAP12 in Ad-LacZ-, Ad-FDAP12-, and Ad-TREM-1 Ig-infected and zymosan A-injected mice. RT-PCR analysis was performed on total RNA extracted from mouse liver. Every liver of four mice in each group represents the same results. B: FLAG-DAP12 and endogenous DAP12 protein expression in F4/80-positive cells from Ad-FDAP12-infected mouse liver 3 days after zymosan A injection. F4/80-positive cells from four Ad-FDAP12-infected mice were collected. Lysates prepared from 1 × 10 6 cells were immunoprecipitated with anti-DAP12 polyclonal antibodies and analyzed by Western blotting using anti-DAP12 polyclonal antibodies or anti-FLAG mAb.
Figure Legend Snippet: A: Gene expression of TREM-1 and FLAG-DAP12 in Ad-LacZ-, Ad-FDAP12-, and Ad-TREM-1 Ig-infected and zymosan A-injected mice. RT-PCR analysis was performed on total RNA extracted from mouse liver. Every liver of four mice in each group represents the same results. B: FLAG-DAP12 and endogenous DAP12 protein expression in F4/80-positive cells from Ad-FDAP12-infected mouse liver 3 days after zymosan A injection. F4/80-positive cells from four Ad-FDAP12-infected mice were collected. Lysates prepared from 1 × 10 6 cells were immunoprecipitated with anti-DAP12 polyclonal antibodies and analyzed by Western blotting using anti-DAP12 polyclonal antibodies or anti-FLAG mAb.

Techniques Used: Expressing, Infection, Injection, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Western Blot

5) Product Images from "Feasibility of Generating Adeno-Associated Virus Packaging Cell Lines Containing Inducible Adenovirus Helper Genes"

Article Title: Feasibility of Generating Adeno-Associated Virus Packaging Cell Lines Containing Inducible Adenovirus Helper Genes

Journal: Journal of Virology

doi: 10.1128/JVI.76.4.1904-1913.2002

Western analysis of capsid gene expression from different cell lines. Various AAV-GFP cell lines were infected with Ad-LacZ in the absence of DOX. The E1A gene was induced by treating the cells with TSA for 12 h. The cell line XX-53-GFP is an AAV packaging cell line lacking the E1 genes. Only wt Ad infection can lead to AAV-GFP production in this cell line. Western analysis was performed with an anticapsid polyclonal antibody which recognizes all three capsid proteins.
Figure Legend Snippet: Western analysis of capsid gene expression from different cell lines. Various AAV-GFP cell lines were infected with Ad-LacZ in the absence of DOX. The E1A gene was induced by treating the cells with TSA for 12 h. The cell line XX-53-GFP is an AAV packaging cell line lacking the E1 genes. Only wt Ad infection can lead to AAV-GFP production in this cell line. Western analysis was performed with an anticapsid polyclonal antibody which recognizes all three capsid proteins.

Techniques Used: Western Blot, Expressing, Infection

Complementation of the E1A-E1B cell lines for Ad-GFP vector production. Plasmid pST-E1AB or pST-E1AB-R was transfected into HeLa-tet-off cells together with a puromycin selection marker plasmid. Puromycin-resistant clones were screened for their ability to complement Ad-GFP production. The representative clones shown here were infected with Ad-GFP (MOI = 5) in the presence or absence of DOX and incubated for 72 h. The yields of Ad-GFP vector were determined by titer determination on 293 cells.
Figure Legend Snippet: Complementation of the E1A-E1B cell lines for Ad-GFP vector production. Plasmid pST-E1AB or pST-E1AB-R was transfected into HeLa-tet-off cells together with a puromycin selection marker plasmid. Puromycin-resistant clones were screened for their ability to complement Ad-GFP production. The representative clones shown here were infected with Ad-GFP (MOI = 5) in the presence or absence of DOX and incubated for 72 h. The yields of Ad-GFP vector were determined by titer determination on 293 cells.

Techniques Used: Plasmid Preparation, Transfection, Selection, Marker, Clone Assay, Infection, Incubation

Construction of inducible Ad gene expression plasmids and AAV plasmids. The Ad type 2 E1A gene (without a promoter) and the E1B gene (with the endogenous promoter) were PCR amplified and cloned into the Eco RV site of plasmid PBI (Clontech), which has a TET-inducible promoter consisting of a minimal CMV promoter and a TRE enhancer. Plasmid pST-E1AB contains the E1A and -B genes in the sense orientation with respect to the TET-inducible promoter, whereas plasmid pST-E1A/B-R has the E1A and -B genes in the reverse orientation. Plasmid pXX2-GFP-Hyg harbors an AAV vector with a GFP reporter gene and a hygromycin resistance gene flanked by two inverted terminal repeats (ITR) of AAV. This plasmid also harbors the AAV viral coding genes Rep and Cap at a different locus. Plasmid pXX6-ZEO contains a zeomycin resistance gene and three Ad genes (VA RNA, E2A, and E4) that can provide helper functions to AAV.
Figure Legend Snippet: Construction of inducible Ad gene expression plasmids and AAV plasmids. The Ad type 2 E1A gene (without a promoter) and the E1B gene (with the endogenous promoter) were PCR amplified and cloned into the Eco RV site of plasmid PBI (Clontech), which has a TET-inducible promoter consisting of a minimal CMV promoter and a TRE enhancer. Plasmid pST-E1AB contains the E1A and -B genes in the sense orientation with respect to the TET-inducible promoter, whereas plasmid pST-E1A/B-R has the E1A and -B genes in the reverse orientation. Plasmid pXX2-GFP-Hyg harbors an AAV vector with a GFP reporter gene and a hygromycin resistance gene flanked by two inverted terminal repeats (ITR) of AAV. This plasmid also harbors the AAV viral coding genes Rep and Cap at a different locus. Plasmid pXX6-ZEO contains a zeomycin resistance gene and three Ad genes (VA RNA, E2A, and E4) that can provide helper functions to AAV.

Techniques Used: Expressing, Polymerase Chain Reaction, Amplification, Clone Assay, Plasmid Preparation

Western analysis of E1A gene expression after TSA and DOX induction. (A) E1A gene expression in three E1 cell lines was examined to see if the gene shutoff can be reversed by TSA. The cells were treated with TSA for 12 h, and E1A gene expression was measured throughout the time course from the beginning of TSA treatment in the absence of DOX repression. 293 cells were used as the positive control for E1A expression without any treatment. (B) E1A gene reactivation and induction in the E1-112 cell line is dependent on both TSA treatment and removal of DOX repression.
Figure Legend Snippet: Western analysis of E1A gene expression after TSA and DOX induction. (A) E1A gene expression in three E1 cell lines was examined to see if the gene shutoff can be reversed by TSA. The cells were treated with TSA for 12 h, and E1A gene expression was measured throughout the time course from the beginning of TSA treatment in the absence of DOX repression. 293 cells were used as the positive control for E1A expression without any treatment. (B) E1A gene reactivation and induction in the E1-112 cell line is dependent on both TSA treatment and removal of DOX repression.

Techniques Used: Western Blot, Expressing, Positive Control

6) Product Images from "The Human Cytomegalovirus Major Immediate-Early Distal Enhancer Region Is Required for Efficient Viral Replication and Immediate-Early Gene Expression"

Article Title: The Human Cytomegalovirus Major Immediate-Early Distal Enhancer Region Is Required for Efficient Viral Replication and Immediate-Early Gene Expression

Journal: Journal of Virology

doi:

Schematic diagram of recombinant HCMVs containing mutations of the MIE regulatory region. HCMV genome and its unique long (U L ) and short (U S ), internal repeat long (IR L ) and short (IR S ), terminal repeat long (TR L ) and short (TR S ), and a-sequence components are depicted. Locations of MIE regulatory region, MIE gene exons 1 to 4 (open boxes), and putative UL128 gene (open box) within U L component are shown. The MIE regulatory region of the WT is composed of proximal promoter (+1 to −64), enhancer (Enh; -65 to -550), unique region (-551 to -749), and modulator (Mod; -750 to -1140). Numerical base positions are assigned relative to start site of MIE RNAs. Recombinant HCMVs rΔ-300/-640 SVgfp , rΔ-300/-1108 SVgfp , rΔ-640/-1108 SVgfp , and r SVgfp were derived from rΔM SVgpt ). rΔ-300/-640 SVgfp , rΔ-300/-1108 SVgfp , and rΔ-640/-1108 SVgfp have deletions from -300 to -640, -300 to -1108, and -640 to -1108, respectively. An enhancerless SV40 early promoter (−138 to +57), gfp ORF, and SV40 early intron and polyadenylation signal were inserted at the site of deletion. r SVgfp has the same insertion at -640 but has no deletion. rΔM SVgpt has a deletion of -640 to -1108 and insertion of the SV40 early transcription unit containing the gpt ORF.
Figure Legend Snippet: Schematic diagram of recombinant HCMVs containing mutations of the MIE regulatory region. HCMV genome and its unique long (U L ) and short (U S ), internal repeat long (IR L ) and short (IR S ), terminal repeat long (TR L ) and short (TR S ), and a-sequence components are depicted. Locations of MIE regulatory region, MIE gene exons 1 to 4 (open boxes), and putative UL128 gene (open box) within U L component are shown. The MIE regulatory region of the WT is composed of proximal promoter (+1 to −64), enhancer (Enh; -65 to -550), unique region (-551 to -749), and modulator (Mod; -750 to -1140). Numerical base positions are assigned relative to start site of MIE RNAs. Recombinant HCMVs rΔ-300/-640 SVgfp , rΔ-300/-1108 SVgfp , rΔ-640/-1108 SVgfp , and r SVgfp were derived from rΔM SVgpt ). rΔ-300/-640 SVgfp , rΔ-300/-1108 SVgfp , and rΔ-640/-1108 SVgfp have deletions from -300 to -640, -300 to -1108, and -640 to -1108, respectively. An enhancerless SV40 early promoter (−138 to +57), gfp ORF, and SV40 early intron and polyadenylation signal were inserted at the site of deletion. r SVgfp has the same insertion at -640 but has no deletion. rΔM SVgpt has a deletion of -640 to -1108 and insertion of the SV40 early transcription unit containing the gpt ORF.

Techniques Used: Recombinant, Sequencing, Derivative Assay

7) Product Images from "Paired-Homeodomain Transcription Factor PAX4 Acts as a Transcriptional Repressor in Early Pancreatic Development"

Article Title: Paired-Homeodomain Transcription Factor PAX4 Acts as a Transcriptional Repressor in Early Pancreatic Development

Journal: Molecular and Cellular Biology

doi:

PAX4 repressor domains. (A) Comparison of PAX4 and PAX6 structure. PD, paired domain; HD, homeodomain. (B) Transient transfections of the indicated cell lines were performed with the chimeric PAX4-GAL4 DBD fusion protein constructs shown and a reporter construct consisting of the GAL4 binding site linked to HSV-TK driving CAT expression (GAL4UAS-TK-CAT). (C) Transient transfections of βTC3 and NIH3T3 cells were performed with a reporter construct consisting of five copies of the GAL4 GAL4 UAS. (D) Transient transfections of αTC1.6 cells were performed with a reporter construct consisting of HSV-TK driving CAT expression without a GAL4 binding site (TK-CAT). (E) Transient transfections of βTC3 cells were performed with a reporter consisting of five GAL4 binding sites linked to the minimal adenovirus E1b promoter driving CAT expression (5XGAL4UAS-E1b-CAT). Note that the lowermost expression construct consists of the PAX6 C-terminal domain fused to the GAL4 DBD. Transfections were performed in duplicate on at least three separate occasions. Relative CAT activity of the GAL4-DBD expression construct alone is set arbitrarily at +1. Graphs show mean ± standard error of the mean.
Figure Legend Snippet: PAX4 repressor domains. (A) Comparison of PAX4 and PAX6 structure. PD, paired domain; HD, homeodomain. (B) Transient transfections of the indicated cell lines were performed with the chimeric PAX4-GAL4 DBD fusion protein constructs shown and a reporter construct consisting of the GAL4 binding site linked to HSV-TK driving CAT expression (GAL4UAS-TK-CAT). (C) Transient transfections of βTC3 and NIH3T3 cells were performed with a reporter construct consisting of five copies of the GAL4 GAL4 UAS. (D) Transient transfections of αTC1.6 cells were performed with a reporter construct consisting of HSV-TK driving CAT expression without a GAL4 binding site (TK-CAT). (E) Transient transfections of βTC3 cells were performed with a reporter consisting of five GAL4 binding sites linked to the minimal adenovirus E1b promoter driving CAT expression (5XGAL4UAS-E1b-CAT). Note that the lowermost expression construct consists of the PAX6 C-terminal domain fused to the GAL4 DBD. Transfections were performed in duplicate on at least three separate occasions. Relative CAT activity of the GAL4-DBD expression construct alone is set arbitrarily at +1. Graphs show mean ± standard error of the mean.

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

8) Product Images from "Multiple Signal Input and Output Domains of the 160-Kilodalton Nuclear Receptor Coactivator Proteins"

Article Title: Multiple Signal Input and Output Domains of the 160-Kilodalton Nuclear Receptor Coactivator Proteins

Journal: Molecular and Cellular Biology

doi:

Binding of GRIP1, SRC-1a, and SRC-1e to AR HBD correlates with coactivator effect on AR AF-2. (A) Alignment and functional domains of p160 coactivators. NID, NR HBD interaction domain; vertical solid bars, NR boxes I, II, and III (LXXLL motifs); NID aux (spotted box), auxiliary domain in GRIP1 required in addition to NR boxes for efficient binding of AR HBD but not TR HBD; AD1 (solid box) and AD2 (striped box), two autonomous activation domains; numbers, amino acids of GRIP1 and SRC-1. The lines between GRIP1 and SRC-1 amino acid numbers indicate alignment of homologous regions. NID aux and AD1 overlap within GRIP1 amino acids 1011 and 1121; SRC-1 lacks NID aux and SRC-1a contains a fourth NR box (IV). (B) Interaction of p160 coactivators with AR HBD in yeast two-hybrid assays. Gal4 AD, fused to the indicated coactivator, was expressed in yeast strain SFY526 along with a Gal4DBD-ARHBD or Gal4DBD-p300 fusion protein; when AR HBD was present, yeast cells were grown in 100 nM DHT. Activation of the integrated β-Gal reporter gene controlled by Gal4 binding sites was determined by measuring β-Gal activity in cell extracts. (C) Enhancement of AR AF-2 function by p160 coactivators. CV-1 cells were transfected with 0.5 μg of pM vector encoding Gal4DBD-ARAF2, 0.5 μg of GK1 reporter gene, and 1.0 μg of pSG5.HA (no CoA) or the same vector encoding the indicated coactivator. Luciferase activity, shown in relative light units (RLU), observed with or without DHT treatment is shown.
Figure Legend Snippet: Binding of GRIP1, SRC-1a, and SRC-1e to AR HBD correlates with coactivator effect on AR AF-2. (A) Alignment and functional domains of p160 coactivators. NID, NR HBD interaction domain; vertical solid bars, NR boxes I, II, and III (LXXLL motifs); NID aux (spotted box), auxiliary domain in GRIP1 required in addition to NR boxes for efficient binding of AR HBD but not TR HBD; AD1 (solid box) and AD2 (striped box), two autonomous activation domains; numbers, amino acids of GRIP1 and SRC-1. The lines between GRIP1 and SRC-1 amino acid numbers indicate alignment of homologous regions. NID aux and AD1 overlap within GRIP1 amino acids 1011 and 1121; SRC-1 lacks NID aux and SRC-1a contains a fourth NR box (IV). (B) Interaction of p160 coactivators with AR HBD in yeast two-hybrid assays. Gal4 AD, fused to the indicated coactivator, was expressed in yeast strain SFY526 along with a Gal4DBD-ARHBD or Gal4DBD-p300 fusion protein; when AR HBD was present, yeast cells were grown in 100 nM DHT. Activation of the integrated β-Gal reporter gene controlled by Gal4 binding sites was determined by measuring β-Gal activity in cell extracts. (C) Enhancement of AR AF-2 function by p160 coactivators. CV-1 cells were transfected with 0.5 μg of pM vector encoding Gal4DBD-ARAF2, 0.5 μg of GK1 reporter gene, and 1.0 μg of pSG5.HA (no CoA) or the same vector encoding the indicated coactivator. Luciferase activity, shown in relative light units (RLU), observed with or without DHT treatment is shown.

Techniques Used: Binding Assay, Functional Assay, Activation Assay, Activity Assay, Transfection, Plasmid Preparation, Luciferase

9) 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

10) Product Images from "Paired-Homeodomain Transcription Factor PAX4 Acts as a Transcriptional Repressor in Early Pancreatic Development"

Article Title: Paired-Homeodomain Transcription Factor PAX4 Acts as a Transcriptional Repressor in Early Pancreatic Development

Journal: Molecular and Cellular Biology

doi:

PAX4 transcriptional repression in fetal pancreatic epithelium. Transient transfections were performed in mouse e11.5 pancreatic epithelium with expression vectors containing the GAL4 DBD coding sequence fused with the PAX4 and PAX6 cDNA fragments shown, the pFOXLuc2.TK.5GAL reporter plasmid, and the pFOXEGFP.CMV internal standard. Transfections were performed in triplicate on individual fetal pancreatic buds. Luciferase mRNA levels were measured by RT-PCR and standardized to EGFP mRNA levels. The graph shows mean ± standard error of the mean.
Figure Legend Snippet: PAX4 transcriptional repression in fetal pancreatic epithelium. Transient transfections were performed in mouse e11.5 pancreatic epithelium with expression vectors containing the GAL4 DBD coding sequence fused with the PAX4 and PAX6 cDNA fragments shown, the pFOXLuc2.TK.5GAL reporter plasmid, and the pFOXEGFP.CMV internal standard. Transfections were performed in triplicate on individual fetal pancreatic buds. Luciferase mRNA levels were measured by RT-PCR and standardized to EGFP mRNA levels. The graph shows mean ± standard error of the mean.

Techniques Used: Transfection, Expressing, Sequencing, Plasmid Preparation, Luciferase, Reverse Transcription Polymerase Chain Reaction

PAX4 repressor domains. (A) Comparison of PAX4 and PAX6 structure. PD, paired domain; HD, homeodomain. (B) Transient transfections of the indicated cell lines were performed with the chimeric PAX4-GAL4 DBD fusion protein constructs shown and a reporter construct consisting of the GAL4 binding site linked to HSV-TK driving CAT expression (GAL4UAS-TK-CAT). (C) Transient transfections of βTC3 and NIH3T3 cells were performed with a reporter construct consisting of five copies of the GAL4 GAL4 UAS. (D) Transient transfections of αTC1.6 cells were performed with a reporter construct consisting of HSV-TK driving CAT expression without a GAL4 binding site (TK-CAT). (E) Transient transfections of βTC3 cells were performed with a reporter consisting of five GAL4 binding sites linked to the minimal adenovirus E1b promoter driving CAT expression (5XGAL4UAS-E1b-CAT). Note that the lowermost expression construct consists of the PAX6 C-terminal domain fused to the GAL4 DBD. Transfections were performed in duplicate on at least three separate occasions. Relative CAT activity of the GAL4-DBD expression construct alone is set arbitrarily at +1. Graphs show mean ± standard error of the mean.
Figure Legend Snippet: PAX4 repressor domains. (A) Comparison of PAX4 and PAX6 structure. PD, paired domain; HD, homeodomain. (B) Transient transfections of the indicated cell lines were performed with the chimeric PAX4-GAL4 DBD fusion protein constructs shown and a reporter construct consisting of the GAL4 binding site linked to HSV-TK driving CAT expression (GAL4UAS-TK-CAT). (C) Transient transfections of βTC3 and NIH3T3 cells were performed with a reporter construct consisting of five copies of the GAL4 GAL4 UAS. (D) Transient transfections of αTC1.6 cells were performed with a reporter construct consisting of HSV-TK driving CAT expression without a GAL4 binding site (TK-CAT). (E) Transient transfections of βTC3 cells were performed with a reporter consisting of five GAL4 binding sites linked to the minimal adenovirus E1b promoter driving CAT expression (5XGAL4UAS-E1b-CAT). Note that the lowermost expression construct consists of the PAX6 C-terminal domain fused to the GAL4 DBD. Transfections were performed in duplicate on at least three separate occasions. Relative CAT activity of the GAL4-DBD expression construct alone is set arbitrarily at +1. Graphs show mean ± standard error of the mean.

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

11) 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

12) Product Images from "Paired-Homeodomain Transcription Factor PAX4 Acts as a Transcriptional Repressor in Early Pancreatic Development"

Article Title: Paired-Homeodomain Transcription Factor PAX4 Acts as a Transcriptional Repressor in Early Pancreatic Development

Journal: Molecular and Cellular Biology

doi:

PAX4 repressor domains. (A) Comparison of PAX4 and PAX6 structure. PD, paired domain; HD, homeodomain. (B) Transient transfections of the indicated cell lines were performed with the chimeric PAX4-GAL4 DBD fusion protein constructs shown and a reporter construct consisting of the GAL4 binding site linked to HSV-TK driving CAT expression (GAL4UAS-TK-CAT). (C) Transient transfections of βTC3 and NIH3T3 cells were performed with a reporter construct consisting of five copies of the GAL4 GAL4 UAS. (D) Transient transfections of αTC1.6 cells were performed with a reporter construct consisting of HSV-TK driving CAT expression without a GAL4 binding site (TK-CAT). (E) Transient transfections of βTC3 cells were performed with a reporter consisting of five GAL4 binding sites linked to the minimal adenovirus E1b promoter driving CAT expression (5XGAL4UAS-E1b-CAT). Note that the lowermost expression construct consists of the PAX6 C-terminal domain fused to the GAL4 DBD. Transfections were performed in duplicate on at least three separate occasions. Relative CAT activity of the GAL4-DBD expression construct alone is set arbitrarily at +1. Graphs show mean ± standard error of the mean.
Figure Legend Snippet: PAX4 repressor domains. (A) Comparison of PAX4 and PAX6 structure. PD, paired domain; HD, homeodomain. (B) Transient transfections of the indicated cell lines were performed with the chimeric PAX4-GAL4 DBD fusion protein constructs shown and a reporter construct consisting of the GAL4 binding site linked to HSV-TK driving CAT expression (GAL4UAS-TK-CAT). (C) Transient transfections of βTC3 and NIH3T3 cells were performed with a reporter construct consisting of five copies of the GAL4 GAL4 UAS. (D) Transient transfections of αTC1.6 cells were performed with a reporter construct consisting of HSV-TK driving CAT expression without a GAL4 binding site (TK-CAT). (E) Transient transfections of βTC3 cells were performed with a reporter consisting of five GAL4 binding sites linked to the minimal adenovirus E1b promoter driving CAT expression (5XGAL4UAS-E1b-CAT). Note that the lowermost expression construct consists of the PAX6 C-terminal domain fused to the GAL4 DBD. Transfections were performed in duplicate on at least three separate occasions. Relative CAT activity of the GAL4-DBD expression construct alone is set arbitrarily at +1. Graphs show mean ± standard error of the mean.

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

13) Product Images from "Adenovirus E1B 55-Kilodalton Protein Targets SMARCAL1 for Degradation during Infection and Modulates Cellular DNA Replication"

Article Title: Adenovirus E1B 55-Kilodalton Protein Targets SMARCAL1 for Degradation during Infection and Modulates Cellular DNA Replication

Journal: Journal of Virology

doi: 10.1128/JVI.00402-19

SMARCAL1 is targeted for degradation during Ad infection. A549 cells were either mock infected or infected with 10 PFU/cell of wt Ad5 or wt Ad12 and harvested at the appropriate times postinfection. (A) Ad5 cell lysates were then subjected to WB for SMARCAL1, p53, E1B-55K, E4orf6, and β-actin. (B) Ad12 cell lysates were subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of more than three independent experiments.
Figure Legend Snippet: SMARCAL1 is targeted for degradation during Ad infection. A549 cells were either mock infected or infected with 10 PFU/cell of wt Ad5 or wt Ad12 and harvested at the appropriate times postinfection. (A) Ad5 cell lysates were then subjected to WB for SMARCAL1, p53, E1B-55K, E4orf6, and β-actin. (B) Ad12 cell lysates were subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of more than three independent experiments.

Techniques Used: Infection, Western Blot

SMARCAL1 is degraded during Ad infection in an E1B-55K/E4orf6- and CRL-dependent manner. (A) A549 cells were either mock infected, infected with wt Ad5, or infected with E1B-55K ( dl 1520), E4orf3 (H5 pm 4150), or E4orf6 (H5 pm 4154) deletion virus. At 24 h and 48 h postinfection, cells were harvested and subjected to WB for SMARCAL1, p53, E1B-55K, E4orf3, E4orf6, and β-actin. (B) A549 cells were either mock infected, infected with wt Ad12, or infected with the E1B-55K ( dl 620) deletion virus. At 24 h and 48 h postinfection, cells were harvested and Western blotted for SMARCAL1, p53, E1B-55K, and β-actin. (C and D) A549 cells were either mock infected or infected with wt Ad5 or wt Ad12 in the absence or presence of 100 nM or 500 nM MLN4924. At 24 h and 48 h postinfection, cells were harvested and subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of three independent experiments.
Figure Legend Snippet: SMARCAL1 is degraded during Ad infection in an E1B-55K/E4orf6- and CRL-dependent manner. (A) A549 cells were either mock infected, infected with wt Ad5, or infected with E1B-55K ( dl 1520), E4orf3 (H5 pm 4150), or E4orf6 (H5 pm 4154) deletion virus. At 24 h and 48 h postinfection, cells were harvested and subjected to WB for SMARCAL1, p53, E1B-55K, E4orf3, E4orf6, and β-actin. (B) A549 cells were either mock infected, infected with wt Ad12, or infected with the E1B-55K ( dl 620) deletion virus. At 24 h and 48 h postinfection, cells were harvested and Western blotted for SMARCAL1, p53, E1B-55K, and β-actin. (C and D) A549 cells were either mock infected or infected with wt Ad5 or wt Ad12 in the absence or presence of 100 nM or 500 nM MLN4924. At 24 h and 48 h postinfection, cells were harvested and subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of three independent experiments.

Techniques Used: Infection, Western Blot

Generation and characterization of tetracycline-inducible Ad5 and Ad12 E1B-55K FlpIn U2OS cells. FlpIn U2OS cells were transfected with Ad5 E1B-55K and Ad12 E1B-55K pcDNA5/FRT/TO plasmids and the recombination plasmid pOG44. Cells were incubated in selection medium containing hygromycin (200 μg/ml). Individual colonies were isolated, expanded, and treated with 0.1 μg/ml doxycycline. Twenty-four h postinduction, cell lysates were harvested, separated by SDS-PAGE, and subjected to WB analysis for Ad5 and Ad12 E1B-55K. WB analyses were also performed to gauge the levels of SMARCAL1, p53, MRE11, and β-actin for Ad5 E1B-55K and Ad12 E1B-55K FlpIn U2OS cells. Data are representative of more than three independent experiments.
Figure Legend Snippet: Generation and characterization of tetracycline-inducible Ad5 and Ad12 E1B-55K FlpIn U2OS cells. FlpIn U2OS cells were transfected with Ad5 E1B-55K and Ad12 E1B-55K pcDNA5/FRT/TO plasmids and the recombination plasmid pOG44. Cells were incubated in selection medium containing hygromycin (200 μg/ml). Individual colonies were isolated, expanded, and treated with 0.1 μg/ml doxycycline. Twenty-four h postinduction, cell lysates were harvested, separated by SDS-PAGE, and subjected to WB analysis for Ad5 and Ad12 E1B-55K. WB analyses were also performed to gauge the levels of SMARCAL1, p53, MRE11, and β-actin for Ad5 E1B-55K and Ad12 E1B-55K FlpIn U2OS cells. Data are representative of more than three independent experiments.

Techniques Used: Transfection, Plasmid Preparation, Incubation, Selection, Isolation, SDS Page, Western Blot

Ad E1B-55K associates with SMARCAL1 in Ad-transformed cells. (A) Ad E1B-55K and SMARCAL1 were immunoprecipitated from Ad5 HEK 293 cells (A) and Ad12 HER2 cells (B) and subjected to WB for E1B-55K and SMARCAL1. IgG, immunoglobulin control IP.
Figure Legend Snippet: Ad E1B-55K associates with SMARCAL1 in Ad-transformed cells. (A) Ad E1B-55K and SMARCAL1 were immunoprecipitated from Ad5 HEK 293 cells (A) and Ad12 HER2 cells (B) and subjected to WB for E1B-55K and SMARCAL1. IgG, immunoglobulin control IP.

Techniques Used: Transformation Assay, Immunoprecipitation, Western Blot

ATR kinase and CDKs promote SMARCAL1 degradation following Ad5 and Ad12 infection. A549 cells were either mock infected or infected with 10 PFU/cell of wt Ad5 (A and C) or wt Ad12 (B and D). Cells were then incubated in the absence or presence of ATR inhibitor (AZD6738 [ATRi], 1 μM; A and B) or ATR and CDK inhibitors (AZD6738, 1 μM and RO-3306 [CDKi], 9 μM; C and D) and harvested at the appropriate times postinfection. Cell lysates were then separated by SDS-PAGE and subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of three independent experiments.
Figure Legend Snippet: ATR kinase and CDKs promote SMARCAL1 degradation following Ad5 and Ad12 infection. A549 cells were either mock infected or infected with 10 PFU/cell of wt Ad5 (A and C) or wt Ad12 (B and D). Cells were then incubated in the absence or presence of ATR inhibitor (AZD6738 [ATRi], 1 μM; A and B) or ATR and CDK inhibitors (AZD6738, 1 μM and RO-3306 [CDKi], 9 μM; C and D) and harvested at the appropriate times postinfection. Cell lysates were then separated by SDS-PAGE and subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of three independent experiments.

Techniques Used: Infection, Incubation, SDS Page, Western Blot

Ad5 and Ad12 E1B-55K modulate cellular DNA replication rates and promote replication fork stalling. Uninduced and doxycycline-induced Ad5 and Ad12 E1B-55K FlpIn U2OS cells were labeled with 25 μM CldU and 250 μM IdU for 20 min each. DNA fiber spreads were then prepared and denatured with 2.5 M HCl. DNA fibers were labeled with the appropriate primary and secondary antibodies and visualized using a Nikon E600 microscope. (A and B) Representative DNA spreads (with or without Ad5 or Ad12 E1B-55K) are shown indicating the mean fork speeds; CldU and IdU fork lengths were quantified and presented as dot plots (± standard deviations [SD]), with the mean fork speed shown as a red bar. n = 3 (total fibers analyzed: Ad5 mock infected, 347; Ad5 E1B-55K, 368; Ad12 mock infected, 370; Ad12 E1B-55K, 364). (C) Percent stalled forks (CldU-only labeled forks) were quantified and presented as a bar chart (±SD). In all instances data presented were subjected to analysis of variance with two-tailed t test: Ad5 E1B-55K CldU tract length relative to the mock CldU tract length, P = 4.8E−20 (***); Ad5 E1B-55K CldU/IdU ratio relative to the mock CldU tract length, P = 9.44E−45 (****); Ad12 E1B-55K CldU tract length relative to the mock CldU tract length, P = 1.29E−32 (****); Ad12 E1B-55K CldU/IdU ratio relative to the mock CldU tract length, P = 6.32E−61 (****); ns, not significant. For significance for stalled forks, Ad5 E1B-55K relative to mock infection, P = 0.009 (**); Ad12 E1B-55K relative mock infection, P = 0.002 (**).
Figure Legend Snippet: Ad5 and Ad12 E1B-55K modulate cellular DNA replication rates and promote replication fork stalling. Uninduced and doxycycline-induced Ad5 and Ad12 E1B-55K FlpIn U2OS cells were labeled with 25 μM CldU and 250 μM IdU for 20 min each. DNA fiber spreads were then prepared and denatured with 2.5 M HCl. DNA fibers were labeled with the appropriate primary and secondary antibodies and visualized using a Nikon E600 microscope. (A and B) Representative DNA spreads (with or without Ad5 or Ad12 E1B-55K) are shown indicating the mean fork speeds; CldU and IdU fork lengths were quantified and presented as dot plots (± standard deviations [SD]), with the mean fork speed shown as a red bar. n = 3 (total fibers analyzed: Ad5 mock infected, 347; Ad5 E1B-55K, 368; Ad12 mock infected, 370; Ad12 E1B-55K, 364). (C) Percent stalled forks (CldU-only labeled forks) were quantified and presented as a bar chart (±SD). In all instances data presented were subjected to analysis of variance with two-tailed t test: Ad5 E1B-55K CldU tract length relative to the mock CldU tract length, P = 4.8E−20 (***); Ad5 E1B-55K CldU/IdU ratio relative to the mock CldU tract length, P = 9.44E−45 (****); Ad12 E1B-55K CldU tract length relative to the mock CldU tract length, P = 1.29E−32 (****); Ad12 E1B-55K CldU/IdU ratio relative to the mock CldU tract length, P = 6.32E−61 (****); ns, not significant. For significance for stalled forks, Ad5 E1B-55K relative to mock infection, P = 0.009 (**); Ad12 E1B-55K relative mock infection, P = 0.002 (**).

Techniques Used: Labeling, Microscopy, Infection, Two Tailed Test

14) Product Images from "Modulation of Hepatic Granulomatous Responses by Transgene Expression of DAP12 or TREM-1-Ig Molecules"

Article Title: Modulation of Hepatic Granulomatous Responses by Transgene Expression of DAP12 or TREM-1-Ig Molecules

Journal: The American Journal of Pathology

doi:

A: Illustration of the mode by which Ad-FDAP12 and Ad-TREM-1 Ig vectors modulate the DAP12-mediated signaling pathway in myeloid lineage cells or monocytes/neutrophils. The membrane-anchored protein FDAP12 derived from Ad-FDAP12 is flagged in the diagram with circular shapes that distinguish from the endogenous DAP12 molecule. The signal through DAP12-associating molecules (TREM family, SIRPβ1, MDL-1) is transmitted to both FDAP12 and endogenous DAP12. TREM-1 Ig, the adenoviral-derived extracellular domain of TREM-1 that was linked to the human Ig Fc portion ( dotted in the diagram), serves as an inhibitor of DAP12 signaling pathway by competing with an as yet unidentified ligand for binding to the TREM-1 molecule. B: Construction of adenovirus vectors. The elements inserted into the adenoviral genome are illustrated in two bars ( top ) for Ad-FDAP12 and Ad-TREM-1 Ig vectors, respectively. The pAxCAwt cosmid vector containing the above insert was co-transfected into 293 cells with restriction enzyme-digested DNA-TPC (Ad genome tagged with 55-kd terminal protein) to generate recombinant adenoviruses. EC, Extracellular domain; CAG promoter, cytomegalovirus enhancer and chicken β-actin promoter; G poly A, rabbit β-globin poly A signal; FLAG, 24 nucleotides coding for eight defined amino acids (DYKDDDDK) serving as a tag; ApR, ampicillin-resistance gene; cos, cos site of λ phage; ori, replication origin.
Figure Legend Snippet: A: Illustration of the mode by which Ad-FDAP12 and Ad-TREM-1 Ig vectors modulate the DAP12-mediated signaling pathway in myeloid lineage cells or monocytes/neutrophils. The membrane-anchored protein FDAP12 derived from Ad-FDAP12 is flagged in the diagram with circular shapes that distinguish from the endogenous DAP12 molecule. The signal through DAP12-associating molecules (TREM family, SIRPβ1, MDL-1) is transmitted to both FDAP12 and endogenous DAP12. TREM-1 Ig, the adenoviral-derived extracellular domain of TREM-1 that was linked to the human Ig Fc portion ( dotted in the diagram), serves as an inhibitor of DAP12 signaling pathway by competing with an as yet unidentified ligand for binding to the TREM-1 molecule. B: Construction of adenovirus vectors. The elements inserted into the adenoviral genome are illustrated in two bars ( top ) for Ad-FDAP12 and Ad-TREM-1 Ig vectors, respectively. The pAxCAwt cosmid vector containing the above insert was co-transfected into 293 cells with restriction enzyme-digested DNA-TPC (Ad genome tagged with 55-kd terminal protein) to generate recombinant adenoviruses. EC, Extracellular domain; CAG promoter, cytomegalovirus enhancer and chicken β-actin promoter; G poly A, rabbit β-globin poly A signal; FLAG, 24 nucleotides coding for eight defined amino acids (DYKDDDDK) serving as a tag; ApR, ampicillin-resistance gene; cos, cos site of λ phage; ori, replication origin.

Techniques Used: Derivative Assay, Binding Assay, Plasmid Preparation, Transfection, Recombinant

Zymosan A-induced hepatic granuloma formation in Ad-LacZ ( A , D )-, Ad-FDAP12 ( B , E )-, and Ad-TREM-1 Ig ( C , F )-infected mice at day 3. Liver sections were subjected to H E staining ( A–C ) and immunostaining for F4/80 ( D–F ). Original magnifications, ×100.
Figure Legend Snippet: Zymosan A-induced hepatic granuloma formation in Ad-LacZ ( A , D )-, Ad-FDAP12 ( B , E )-, and Ad-TREM-1 Ig ( C , F )-infected mice at day 3. Liver sections were subjected to H E staining ( A–C ) and immunostaining for F4/80 ( D–F ). Original magnifications, ×100.

Techniques Used: Infection, Mouse Assay, Staining, Immunostaining

Zymosan A-induced hepatic granuloma formation in Ad-LacZ ( A , D )-, Ad-FDAP12 ( B , E )-, and Ad-TREM-1 Ig ( C , F )-infected mice at day 5. Liver sections were subjected to H E staining ( A–C ) and immunostaining for F4/80 ( D–F ). Original magnifications, ×100.
Figure Legend Snippet: Zymosan A-induced hepatic granuloma formation in Ad-LacZ ( A , D )-, Ad-FDAP12 ( B , E )-, and Ad-TREM-1 Ig ( C , F )-infected mice at day 5. Liver sections were subjected to H E staining ( A–C ) and immunostaining for F4/80 ( D–F ). Original magnifications, ×100.

Techniques Used: Infection, Mouse Assay, Staining, Immunostaining

Zymosan A-induced hepatic granuloma formation in Ad-LacZ ( A , D )-, Ad-FDAP12 ( B , E )-, and Ad-TREM-1 Ig ( C , F )-infected mice at day 10. Liver sections were subjected to H E staining ( A–C ) and immunostaining for F4/80 ( D–F ). Original magnifications, ×100.
Figure Legend Snippet: Zymosan A-induced hepatic granuloma formation in Ad-LacZ ( A , D )-, Ad-FDAP12 ( B , E )-, and Ad-TREM-1 Ig ( C , F )-infected mice at day 10. Liver sections were subjected to H E staining ( A–C ) and immunostaining for F4/80 ( D–F ). Original magnifications, ×100.

Techniques Used: Infection, Mouse Assay, Staining, Immunostaining

Zymosan A-induced hepatic granuloma formation in Ad-DAP12-infected mice was augmented by anti-FLAG mAb. Twenty-four hours after zymosan A injection, mice were infected with Ad-FDAP12 plus isotype control mouse IgG1 ( A , D ), Ad-FDAP12 plus anti-FLAG mAb ( B , E ), and Ad-FDAP12 plus Ad-TREM-1 Ig plus anti-FLAG mAb ( C , F ). At day 7, mice were killed and liver sections were subjected to H E staining ( A–C ) and immunostaining for F4/80 ( D–F ). Original magnifications, ×100.
Figure Legend Snippet: Zymosan A-induced hepatic granuloma formation in Ad-DAP12-infected mice was augmented by anti-FLAG mAb. Twenty-four hours after zymosan A injection, mice were infected with Ad-FDAP12 plus isotype control mouse IgG1 ( A , D ), Ad-FDAP12 plus anti-FLAG mAb ( B , E ), and Ad-FDAP12 plus Ad-TREM-1 Ig plus anti-FLAG mAb ( C , F ). At day 7, mice were killed and liver sections were subjected to H E staining ( A–C ) and immunostaining for F4/80 ( D–F ). Original magnifications, ×100.

Techniques Used: Infection, Mouse Assay, Injection, Staining, Immunostaining

Higher magnification of zymosan A-induced hepatic granuloma. A–D: Ad-LacZ-treated mice; E–H: Ad-FDAP12-treated mice; and I–L B Ad-TREM-1 Ig-treated mice. PMN, Polymorphonuclear leukocyte; Mφ, macrophage; LC, lymphocyte; Epi, epithelioid cell. Liver sections were subjected to H E staining and observed at a high magnification of ×400.
Figure Legend Snippet: Higher magnification of zymosan A-induced hepatic granuloma. A–D: Ad-LacZ-treated mice; E–H: Ad-FDAP12-treated mice; and I–L B Ad-TREM-1 Ig-treated mice. PMN, Polymorphonuclear leukocyte; Mφ, macrophage; LC, lymphocyte; Epi, epithelioid cell. Liver sections were subjected to H E staining and observed at a high magnification of ×400.

Techniques Used: Mouse Assay, Staining

Enumeration of granulomas in the liver throughout time in Ad-LacZ-, Ad-FDAP12-, and Ad-TREM-1 Ig-infected mice ( n = 4). The results were analyzed using one-way analysis of variance and post hoc test. Granulomas were calculated in randomly selected 10 fields at an original magnification of ×200.
Figure Legend Snippet: Enumeration of granulomas in the liver throughout time in Ad-LacZ-, Ad-FDAP12-, and Ad-TREM-1 Ig-infected mice ( n = 4). The results were analyzed using one-way analysis of variance and post hoc test. Granulomas were calculated in randomly selected 10 fields at an original magnification of ×200.

Techniques Used: Infection, Mouse Assay

Zymosan A-induced hepatic granuloma formation in Ad-LacZ ( A , D )-, Ad-FDAP12 ( B , E )-, and Ad-TREM-1 Ig ( C , F )-infected mice at day 7. Liver sections were subjected to H E staining ( A–C ) and immunostaining for F4/80 ( D–F ). Original magnifications, ×100.
Figure Legend Snippet: Zymosan A-induced hepatic granuloma formation in Ad-LacZ ( A , D )-, Ad-FDAP12 ( B , E )-, and Ad-TREM-1 Ig ( C , F )-infected mice at day 7. Liver sections were subjected to H E staining ( A–C ) and immunostaining for F4/80 ( D–F ). Original magnifications, ×100.

Techniques Used: Infection, Mouse Assay, Staining, Immunostaining

A: Gene expression of TREM-1 and FLAG-DAP12 in Ad-LacZ-, Ad-FDAP12-, and Ad-TREM-1 Ig-infected and zymosan A-injected mice. RT-PCR analysis was performed on total RNA extracted from mouse liver. Every liver of four mice in each group represents the same results. B: FLAG-DAP12 and endogenous DAP12 protein expression in F4/80-positive cells from Ad-FDAP12-infected mouse liver 3 days after zymosan A injection. F4/80-positive cells from four Ad-FDAP12-infected mice were collected. Lysates prepared from 1 × 10 6 cells were immunoprecipitated with anti-DAP12 polyclonal antibodies and analyzed by Western blotting using anti-DAP12 polyclonal antibodies or anti-FLAG mAb.
Figure Legend Snippet: A: Gene expression of TREM-1 and FLAG-DAP12 in Ad-LacZ-, Ad-FDAP12-, and Ad-TREM-1 Ig-infected and zymosan A-injected mice. RT-PCR analysis was performed on total RNA extracted from mouse liver. Every liver of four mice in each group represents the same results. B: FLAG-DAP12 and endogenous DAP12 protein expression in F4/80-positive cells from Ad-FDAP12-infected mouse liver 3 days after zymosan A injection. F4/80-positive cells from four Ad-FDAP12-infected mice were collected. Lysates prepared from 1 × 10 6 cells were immunoprecipitated with anti-DAP12 polyclonal antibodies and analyzed by Western blotting using anti-DAP12 polyclonal antibodies or anti-FLAG mAb.

Techniques Used: Expressing, Infection, Injection, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Western Blot

15) Product Images from "Differential Roles of Smad1 and p38 Kinase in Regulation of Peroxisome Proliferator-activating Receptor ? during Bone Morphogenetic Protein 2-induced Adipogenesis"

Article Title: Differential Roles of Smad1 and p38 Kinase in Regulation of Peroxisome Proliferator-activating Receptor ? during Bone Morphogenetic Protein 2-induced Adipogenesis

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E02-06-0356

(A) Time-dependent activation of p38 kinase by BMP2 in C3H10T1/2. C3H10T1/2 cells were serum starved in 0.2% fetal calf serum-DMEM for 16 h. The cells were then stimulated with BMP2 (300 ng/ml) for 5, 10, 15, 30, or 60 min and lysed, and the cell lysates were determined by immunoblotting with anti-phospho-p38 kinase (top) and anti-p38 kinase antibodies (bottom). (B) Activation of p38 kinase in C3H10T1/2 and its inhibition by SB203580. C3H10T1/2 cells were serum starved in 0.2% fetal calf serum-DMEM for 12 h. The cells were further incubated with or without p38 kinase inhibitor SB203580 (10 μM), for 4 h. The cells were then stimulated with BMP2 (300 ng/ml) for 10 min and lysed, and the cell lysates were determined by immunoblotting with anti-phospho-p38 kinase (top) and anti-p38 kinase antibodies (bottom). (C) SB203580 did not affect the phosphorylation of Smad1. C3H10T1/2 cells were serum starved in 0.2% fetal calf serum-DMEM for 12 h. The cells were further incubated with or without SB203580 (10 μM) for 4 h. The cells were then stimulated with BMP2 (300 ng/ml) for 10 min and lysed. The cell lysates were immunoprecipitated with anti-Smad1 and immunoblotting with anti-phosphoserine (top) or anti-Smad1 antibodies (bottom). (D) SB203580 inhibited transcriptional activity of PPARγ induced by BMP2. C3H10T1/2 cells were transfected into together with PPARγ (0.1 μg) and RXR (0.1 μg) expression vectors, and PPRE-Luc (0.1 μg) and TK-renilla (0.1 μg) reporter constructs, and pcDNA3 (1.6 μg). Twelve hours after transfection, cells were incubated with or without SB203580 (10 μM) for 2 d. At the end of the culture, cells were lysed and the luciferase activity was measured and normalized by determining renilla luciferase activity as described in the text. Data are shown as mean ± SD. (E) SB203580 did not affect the expression of PPARγ. C3H10T1/2 cells were incubated with BMP2 (300 ng/ml), SB203580 (10 μM), or both for 3 d. The cells were lysed, and the expression of PPARγ was determined by immunoblotting with anti-PPARγ monoclonal antibody, followed by immunoprecipitation with anti-PPARγ polyclonal antibody (top). The lysates were also determined by immunoblotting with anti-β-actin antibody (bottom).
Figure Legend Snippet: (A) Time-dependent activation of p38 kinase by BMP2 in C3H10T1/2. C3H10T1/2 cells were serum starved in 0.2% fetal calf serum-DMEM for 16 h. The cells were then stimulated with BMP2 (300 ng/ml) for 5, 10, 15, 30, or 60 min and lysed, and the cell lysates were determined by immunoblotting with anti-phospho-p38 kinase (top) and anti-p38 kinase antibodies (bottom). (B) Activation of p38 kinase in C3H10T1/2 and its inhibition by SB203580. C3H10T1/2 cells were serum starved in 0.2% fetal calf serum-DMEM for 12 h. The cells were further incubated with or without p38 kinase inhibitor SB203580 (10 μM), for 4 h. The cells were then stimulated with BMP2 (300 ng/ml) for 10 min and lysed, and the cell lysates were determined by immunoblotting with anti-phospho-p38 kinase (top) and anti-p38 kinase antibodies (bottom). (C) SB203580 did not affect the phosphorylation of Smad1. C3H10T1/2 cells were serum starved in 0.2% fetal calf serum-DMEM for 12 h. The cells were further incubated with or without SB203580 (10 μM) for 4 h. The cells were then stimulated with BMP2 (300 ng/ml) for 10 min and lysed. The cell lysates were immunoprecipitated with anti-Smad1 and immunoblotting with anti-phosphoserine (top) or anti-Smad1 antibodies (bottom). (D) SB203580 inhibited transcriptional activity of PPARγ induced by BMP2. C3H10T1/2 cells were transfected into together with PPARγ (0.1 μg) and RXR (0.1 μg) expression vectors, and PPRE-Luc (0.1 μg) and TK-renilla (0.1 μg) reporter constructs, and pcDNA3 (1.6 μg). Twelve hours after transfection, cells were incubated with or without SB203580 (10 μM) for 2 d. At the end of the culture, cells were lysed and the luciferase activity was measured and normalized by determining renilla luciferase activity as described in the text. Data are shown as mean ± SD. (E) SB203580 did not affect the expression of PPARγ. C3H10T1/2 cells were incubated with BMP2 (300 ng/ml), SB203580 (10 μM), or both for 3 d. The cells were lysed, and the expression of PPARγ was determined by immunoblotting with anti-PPARγ monoclonal antibody, followed by immunoprecipitation with anti-PPARγ polyclonal antibody (top). The lysates were also determined by immunoblotting with anti-β-actin antibody (bottom).

Techniques Used: Activation Assay, Inhibition, Incubation, Immunoprecipitation, Activity Assay, Transfection, Expressing, Construct, Luciferase

Schematic model of BMP2-induced adipogenesis. Activation of Smad1 is essential for induction of PPARγ expression, which is critical for adipogenic effects of BMP2. In contrast, activation of p38 kinase, presumably through MKK3 and MKK6, plays roles in the up-regulation of transcriptional activity of PPARγ.
Figure Legend Snippet: Schematic model of BMP2-induced adipogenesis. Activation of Smad1 is essential for induction of PPARγ expression, which is critical for adipogenic effects of BMP2. In contrast, activation of p38 kinase, presumably through MKK3 and MKK6, plays roles in the up-regulation of transcriptional activity of PPARγ.

Techniques Used: Activation Assay, Expressing, Activity Assay

(A) Dominant-negative PPARγ blocked BMP2-induced adipogenesis of C3H10T1/2 cells. C3H10T1/2 cells infected with control or dominant-negative PPARγ adenovirus at 100 multiplicity of infection were incubated with or without BMP2 (300 ng/ml) for 10 d. The cells were stained with Oil Red O, photographed under a microscope, and the areas stained with Oil Red O in the cells were assessed. (B) Overexpression of PPARγ-induced adipogenesis. C3H10T1/2 cells infected with control or PPARγ adenovirus at 100 multiplicity of infection were incubated with or without BMP2 (300 ng/ml) for 7 d. The cells were stained with Oil Red O, photographed under a microscope, and the areas stained with Oil Red O in the cells were assessed.
Figure Legend Snippet: (A) Dominant-negative PPARγ blocked BMP2-induced adipogenesis of C3H10T1/2 cells. C3H10T1/2 cells infected with control or dominant-negative PPARγ adenovirus at 100 multiplicity of infection were incubated with or without BMP2 (300 ng/ml) for 10 d. The cells were stained with Oil Red O, photographed under a microscope, and the areas stained with Oil Red O in the cells were assessed. (B) Overexpression of PPARγ-induced adipogenesis. C3H10T1/2 cells infected with control or PPARγ adenovirus at 100 multiplicity of infection were incubated with or without BMP2 (300 ng/ml) for 7 d. The cells were stained with Oil Red O, photographed under a microscope, and the areas stained with Oil Red O in the cells were assessed.

Techniques Used: Dominant Negative Mutation, Infection, Incubation, Staining, Microscopy, Over Expression

Overexpression of Smad6 inhibited activation of Smad1 (A), BMP2-regulated induction of PPARγ (B), and adipogenesis (C and D). (A) C3H10T1/2 cells infected with control or Flag-Smad6 adenovirus at 100 multiplicity of infection were starved with 0.2% fetal calf serum-DMEM for 16 h and then stimulated with or without BMP2 (300 ng/ml) for 10 min and lysed. The cell lysates were immunprecipitated with anti-Smad1 antibody and immunoblotted with anti-phsophoserine antibody (top). The amount of Smad1 in the immunoprecipitates was determined by immunoblotting with anti-Smad1 (second panel). The lysates were also determined by immunoblotting with antiphsopho-p38 (third panel) or antip38 (forth panel) antibodies. The expression of Flag-Smad6 was determined by immunoblotting with anti-Flag antibody (bottom). (B) C3H10T1/2 cells infected with control or Flag-Smad6 adenovirus at 100 multiplicity of infection were incubated with or without BMP2 (300 ng/ml) for 4 d and lysed. The lysates were determined by immunoblotting with anti-PPARγ mAb followed by immunoprecipitation with anti-PPARγ polyclonal antibody (top). The lysates were also determined by immunoblotting with anti-Flag (middle) or anti-β-actin antibodies (bottom). (C and D) 10T1/2 cells infected with control or Flag-Smad6 adenovirus at 100 multiplicity of infection were incubated with or without BMP2 (300 ng/ml) for 10 d. The cells were stained with Oil Red O, photographed under a microscope, and the areas stained with Oil Red O in the cells were assessed.
Figure Legend Snippet: Overexpression of Smad6 inhibited activation of Smad1 (A), BMP2-regulated induction of PPARγ (B), and adipogenesis (C and D). (A) C3H10T1/2 cells infected with control or Flag-Smad6 adenovirus at 100 multiplicity of infection were starved with 0.2% fetal calf serum-DMEM for 16 h and then stimulated with or without BMP2 (300 ng/ml) for 10 min and lysed. The cell lysates were immunprecipitated with anti-Smad1 antibody and immunoblotted with anti-phsophoserine antibody (top). The amount of Smad1 in the immunoprecipitates was determined by immunoblotting with anti-Smad1 (second panel). The lysates were also determined by immunoblotting with antiphsopho-p38 (third panel) or antip38 (forth panel) antibodies. The expression of Flag-Smad6 was determined by immunoblotting with anti-Flag antibody (bottom). (B) C3H10T1/2 cells infected with control or Flag-Smad6 adenovirus at 100 multiplicity of infection were incubated with or without BMP2 (300 ng/ml) for 4 d and lysed. The lysates were determined by immunoblotting with anti-PPARγ mAb followed by immunoprecipitation with anti-PPARγ polyclonal antibody (top). The lysates were also determined by immunoblotting with anti-Flag (middle) or anti-β-actin antibodies (bottom). (C and D) 10T1/2 cells infected with control or Flag-Smad6 adenovirus at 100 multiplicity of infection were incubated with or without BMP2 (300 ng/ml) for 10 d. The cells were stained with Oil Red O, photographed under a microscope, and the areas stained with Oil Red O in the cells were assessed.

Techniques Used: Over Expression, Activation Assay, Infection, Expressing, Incubation, Immunoprecipitation, Staining, Microscopy

(A) BMP2 up-regulated the transcriptional activity of PPARγ in C3H10T1/2 cells. Left, pcDNA3 (control) (1.6 μg) or Smad6 expression vector (1.6 μg) was transfected into C3H10T1/2 cells together with PPARγ (0.1 μg) and RXR (0.1 μg) expression vectors and PPRE-Luc (0.1 μg) and TK-renilla (0.1 μg) reporter constructs. Twelve hours after transfection, cells were incubated with or without BMP2 (300 ng/ml) for 2 d. At the end of the culture, cells were lysed and the luciferase activity was measured and normalized by determining renilla luciferase activity as described in the text. Data were shown as mean ± SD. Right, expression of PPARγ and Smad6 were monitored by immunoblotting with anti-PPARγ (top) and anti-Flag (middle) antibodies in the same set of transfection experiments. The lysates were also determined by immunoblotting with anti-β-actin antibody (bottom). (B) Introduction of PPARγ rescued the adipogenesis blocked by Smad6. C3H10T1/2 cells were infected with Flag-Smad6 (100 multiplicity of infection) and/or PPARγ (100 multiplicity of infection) adenoviruses and incubated with or without BMP2 (300 ng/ml) for 7 d. The cells were stained with Oil Red O, photographed under a microscope, and the areas stained with Oil Red O in the cells were assessed.
Figure Legend Snippet: (A) BMP2 up-regulated the transcriptional activity of PPARγ in C3H10T1/2 cells. Left, pcDNA3 (control) (1.6 μg) or Smad6 expression vector (1.6 μg) was transfected into C3H10T1/2 cells together with PPARγ (0.1 μg) and RXR (0.1 μg) expression vectors and PPRE-Luc (0.1 μg) and TK-renilla (0.1 μg) reporter constructs. Twelve hours after transfection, cells were incubated with or without BMP2 (300 ng/ml) for 2 d. At the end of the culture, cells were lysed and the luciferase activity was measured and normalized by determining renilla luciferase activity as described in the text. Data were shown as mean ± SD. Right, expression of PPARγ and Smad6 were monitored by immunoblotting with anti-PPARγ (top) and anti-Flag (middle) antibodies in the same set of transfection experiments. The lysates were also determined by immunoblotting with anti-β-actin antibody (bottom). (B) Introduction of PPARγ rescued the adipogenesis blocked by Smad6. C3H10T1/2 cells were infected with Flag-Smad6 (100 multiplicity of infection) and/or PPARγ (100 multiplicity of infection) adenoviruses and incubated with or without BMP2 (300 ng/ml) for 7 d. The cells were stained with Oil Red O, photographed under a microscope, and the areas stained with Oil Red O in the cells were assessed.

Techniques Used: Activity Assay, Expressing, Plasmid Preparation, Transfection, Construct, Incubation, Luciferase, Infection, Staining, Microscopy

(A) Overexpression of TAK1- and TAB1-induced activation of p38 kinase in C3H10T1/2 cells. C3H10T1/2 cells were transfected with pcDNA3 vector or both TAK1 and TAB1 expression constructs. Twenty hours after transfection, the cells were serum starved for 16 h and lysed. The cell lysates were determined by immunoblotting with anti-phospho-p38 kinase (top) or anti-p38 kinase (bottom) antibodies. (B) Overexpression of TAK1 and TAB1 up-regulated transcriptional activity of PPARγ. pcDNA3 (control) (1.6 μg) or both TAK1 (0.8 μg) and TAB1 (0.8 μg) expression vectors (1.6 μg) were transfected into C3H10T1/2 cells together with PPARγ (0.1 μg) and RXR (0.1 μg) expression vectors and PPRE-Luc (0.1 μg) and TK-renilla (0.1 μg) reporter constructs. Twelve hours after transfection, cells were incubated with or without SB203580 (10 μM) for 2 d. At the end of the culture, cells were lysed and the luciferase activity was measured and normalized by determining renilla luciferase activity as described in the text. Data were shown as mean ± SD. (C) Dominant-negative MKK3 inhibited BMP2-induced adipogenesis in C3H10T1/2 cells. C3H10T1/2 cells infected with control or dominant-negative MKK3 adenovirus at 100 multiplicity of infection were incubated with or without BMP2 (300 ng/ml). The cells were stained with Oil Red O, photographed under a microscope, and the areas stained with Oil Red O in the cells were assessed. (D) SB203580 inhibited BMP2-induced adipogenesis. C3H10T1/2 cells were incubated with BMP2 (300 ng/ml), SB203580 (10 μM), or both for 10 d. The cells were stained with Oil Red O, photographed under a microscope, and the areas stained with Oil Red O in the cells were assessed.
Figure Legend Snippet: (A) Overexpression of TAK1- and TAB1-induced activation of p38 kinase in C3H10T1/2 cells. C3H10T1/2 cells were transfected with pcDNA3 vector or both TAK1 and TAB1 expression constructs. Twenty hours after transfection, the cells were serum starved for 16 h and lysed. The cell lysates were determined by immunoblotting with anti-phospho-p38 kinase (top) or anti-p38 kinase (bottom) antibodies. (B) Overexpression of TAK1 and TAB1 up-regulated transcriptional activity of PPARγ. pcDNA3 (control) (1.6 μg) or both TAK1 (0.8 μg) and TAB1 (0.8 μg) expression vectors (1.6 μg) were transfected into C3H10T1/2 cells together with PPARγ (0.1 μg) and RXR (0.1 μg) expression vectors and PPRE-Luc (0.1 μg) and TK-renilla (0.1 μg) reporter constructs. Twelve hours after transfection, cells were incubated with or without SB203580 (10 μM) for 2 d. At the end of the culture, cells were lysed and the luciferase activity was measured and normalized by determining renilla luciferase activity as described in the text. Data were shown as mean ± SD. (C) Dominant-negative MKK3 inhibited BMP2-induced adipogenesis in C3H10T1/2 cells. C3H10T1/2 cells infected with control or dominant-negative MKK3 adenovirus at 100 multiplicity of infection were incubated with or without BMP2 (300 ng/ml). The cells were stained with Oil Red O, photographed under a microscope, and the areas stained with Oil Red O in the cells were assessed. (D) SB203580 inhibited BMP2-induced adipogenesis. C3H10T1/2 cells were incubated with BMP2 (300 ng/ml), SB203580 (10 μM), or both for 10 d. The cells were stained with Oil Red O, photographed under a microscope, and the areas stained with Oil Red O in the cells were assessed.

Techniques Used: Over Expression, Activation Assay, Transfection, Plasmid Preparation, Expressing, Construct, Activity Assay, Incubation, Luciferase, Dominant Negative Mutation, Infection, Staining, Microscopy

BMP2 induced adipogenesis in C3H10T1/2 cells. C3H10T1/2 cells were cultured with BMP2 (300 ng/ml), troglitazone (10 −6 M), or both for 10 d. After 10 d, the cells were stained with Oil Red O, photographed under a microscope (A), and the area stained with Oil Red O in the cells was assessed as described in the text (B). (C and D) Treatment of BMP2 initiated the adipogenic effects of troglitazone in C3H10T1/2 cells. C3H10T1/2 cells precultured with or without BMP2 for 3 d (300 ng/ml) were incubated with or without troglitazone (10 −6 M) for 4 d. The cells were stained with Oil Red O, photographed under a microscope (C), and the areas stained with Oil Red O in the cells were assessed (D). (E) BMP2 induced expression of PPARγ in C3H10T1/2 cells. C3H10T1/2 cells were untransfected (lanes 1 and 2) or transfected with pcDNA3 (lane 3) or PPARγ expression vector (lane 4). Twelve hours after transfection, the cells were incubated with (lane 2) or without (lanes 1, 3, and 4) BMP2 for 3 d and lysed. The cell lysates were immunoprecipitated with anti-PPARγ polyclonal antibody and immunoblotted with anti-PPARγ mAb (top). The lysates were also determined by immunoblotting with anti-β-actin antibody (bottom). (F) BMP2 induced expression of PPARγ mRNA in a time-dependent manner in C3H10T1/2 cells. Total RNA was isolated from C3H10T1/2 cells that were incubated with or without BMP2 for 3, 6, 12, or 24 h and subjected to RT-PCR experiments by using specific primers for PPARγ (top) or β-actin (bottom).
Figure Legend Snippet: BMP2 induced adipogenesis in C3H10T1/2 cells. C3H10T1/2 cells were cultured with BMP2 (300 ng/ml), troglitazone (10 −6 M), or both for 10 d. After 10 d, the cells were stained with Oil Red O, photographed under a microscope (A), and the area stained with Oil Red O in the cells was assessed as described in the text (B). (C and D) Treatment of BMP2 initiated the adipogenic effects of troglitazone in C3H10T1/2 cells. C3H10T1/2 cells precultured with or without BMP2 for 3 d (300 ng/ml) were incubated with or without troglitazone (10 −6 M) for 4 d. The cells were stained with Oil Red O, photographed under a microscope (C), and the areas stained with Oil Red O in the cells were assessed (D). (E) BMP2 induced expression of PPARγ in C3H10T1/2 cells. C3H10T1/2 cells were untransfected (lanes 1 and 2) or transfected with pcDNA3 (lane 3) or PPARγ expression vector (lane 4). Twelve hours after transfection, the cells were incubated with (lane 2) or without (lanes 1, 3, and 4) BMP2 for 3 d and lysed. The cell lysates were immunoprecipitated with anti-PPARγ polyclonal antibody and immunoblotted with anti-PPARγ mAb (top). The lysates were also determined by immunoblotting with anti-β-actin antibody (bottom). (F) BMP2 induced expression of PPARγ mRNA in a time-dependent manner in C3H10T1/2 cells. Total RNA was isolated from C3H10T1/2 cells that were incubated with or without BMP2 for 3, 6, 12, or 24 h and subjected to RT-PCR experiments by using specific primers for PPARγ (top) or β-actin (bottom).

Techniques Used: Cell Culture, Staining, Microscopy, Incubation, Expressing, Transfection, Plasmid Preparation, Immunoprecipitation, Isolation, Reverse Transcription Polymerase Chain Reaction

16) 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

17) Product Images from "Adenovirus E1B 55-Kilodalton Protein Targets SMARCAL1 for Degradation during Infection and Modulates Cellular DNA Replication"

Article Title: Adenovirus E1B 55-Kilodalton Protein Targets SMARCAL1 for Degradation during Infection and Modulates Cellular DNA Replication

Journal: Journal of Virology

doi: 10.1128/JVI.00402-19

SMARCAL1 is targeted for degradation during Ad infection. A549 cells were either mock infected or infected with 10 PFU/cell of wt Ad5 or wt Ad12 and harvested at the appropriate times postinfection. (A) Ad5 cell lysates were then subjected to WB for SMARCAL1, p53, E1B-55K, E4orf6, and β-actin. (B) Ad12 cell lysates were subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of more than three independent experiments.
Figure Legend Snippet: SMARCAL1 is targeted for degradation during Ad infection. A549 cells were either mock infected or infected with 10 PFU/cell of wt Ad5 or wt Ad12 and harvested at the appropriate times postinfection. (A) Ad5 cell lysates were then subjected to WB for SMARCAL1, p53, E1B-55K, E4orf6, and β-actin. (B) Ad12 cell lysates were subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of more than three independent experiments.

Techniques Used: Infection, Western Blot

SMARCAL1 is degraded during Ad infection in an E1B-55K/E4orf6- and CRL-dependent manner. (A) A549 cells were either mock infected, infected with wt Ad5, or infected with E1B-55K ( dl 1520), E4orf3 (H5 pm 4150), or E4orf6 (H5 pm 4154) deletion virus. At 24 h and 48 h postinfection, cells were harvested and subjected to WB for SMARCAL1, p53, E1B-55K, E4orf3, E4orf6, and β-actin. (B) A549 cells were either mock infected, infected with wt Ad12, or infected with the E1B-55K ( dl 620) deletion virus. At 24 h and 48 h postinfection, cells were harvested and Western blotted for SMARCAL1, p53, E1B-55K, and β-actin. (C and D) A549 cells were either mock infected or infected with wt Ad5 or wt Ad12 in the absence or presence of 100 nM or 500 nM MLN4924. At 24 h and 48 h postinfection, cells were harvested and subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of three independent experiments.
Figure Legend Snippet: SMARCAL1 is degraded during Ad infection in an E1B-55K/E4orf6- and CRL-dependent manner. (A) A549 cells were either mock infected, infected with wt Ad5, or infected with E1B-55K ( dl 1520), E4orf3 (H5 pm 4150), or E4orf6 (H5 pm 4154) deletion virus. At 24 h and 48 h postinfection, cells were harvested and subjected to WB for SMARCAL1, p53, E1B-55K, E4orf3, E4orf6, and β-actin. (B) A549 cells were either mock infected, infected with wt Ad12, or infected with the E1B-55K ( dl 620) deletion virus. At 24 h and 48 h postinfection, cells were harvested and Western blotted for SMARCAL1, p53, E1B-55K, and β-actin. (C and D) A549 cells were either mock infected or infected with wt Ad5 or wt Ad12 in the absence or presence of 100 nM or 500 nM MLN4924. At 24 h and 48 h postinfection, cells were harvested and subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of three independent experiments.

Techniques Used: Infection, Western Blot

Generation and characterization of tetracycline-inducible Ad5 and Ad12 E1B-55K FlpIn U2OS cells. FlpIn U2OS cells were transfected with Ad5 E1B-55K and Ad12 E1B-55K pcDNA5/FRT/TO plasmids and the recombination plasmid pOG44. Cells were incubated in selection medium containing hygromycin (200 μg/ml). Individual colonies were isolated, expanded, and treated with 0.1 μg/ml doxycycline. Twenty-four h postinduction, cell lysates were harvested, separated by SDS-PAGE, and subjected to WB analysis for Ad5 and Ad12 E1B-55K. WB analyses were also performed to gauge the levels of SMARCAL1, p53, MRE11, and β-actin for Ad5 E1B-55K and Ad12 E1B-55K FlpIn U2OS cells. Data are representative of more than three independent experiments.
Figure Legend Snippet: Generation and characterization of tetracycline-inducible Ad5 and Ad12 E1B-55K FlpIn U2OS cells. FlpIn U2OS cells were transfected with Ad5 E1B-55K and Ad12 E1B-55K pcDNA5/FRT/TO plasmids and the recombination plasmid pOG44. Cells were incubated in selection medium containing hygromycin (200 μg/ml). Individual colonies were isolated, expanded, and treated with 0.1 μg/ml doxycycline. Twenty-four h postinduction, cell lysates were harvested, separated by SDS-PAGE, and subjected to WB analysis for Ad5 and Ad12 E1B-55K. WB analyses were also performed to gauge the levels of SMARCAL1, p53, MRE11, and β-actin for Ad5 E1B-55K and Ad12 E1B-55K FlpIn U2OS cells. Data are representative of more than three independent experiments.

Techniques Used: Transfection, Plasmid Preparation, Incubation, Selection, Isolation, SDS Page, Western Blot

SMARCAL1 is recruited to VRCs in an RPA-dependent and ATR- and CDK-dependent manner. (A) Microscopic images depicting the cellular localization of wt GFP-SMARCAL1, GFP-SMARCAL1-ΔP, and GFP-SMARCAL1-ΔRPA in mock-infected (i to iii), wt Ad5-infected (iv to vi), or wt Ad12-infected cells (vii to ix) 18 h postinfection. (B) Bar graph (± SEM) showing the percentage of GFP-labeled cells that are recruited to VRCs following Ad5 or Ad12 infection. n  = 3 (300 cells per experiment, 900 cells in total). Only those cells that exhibited clear GFP-SMARCAL1 structures in Ad-infected cells, comparable to the known architecture of VRCs at different stages of infection, were counted as VRC positive. Data presented were subjected to analysis of variance with a two-tailed t test. For significance testing for difference in recruitment of GFP-SMARCAL1-ΔP to VRCs relative to that of the wt GFP-SMARCAL1 following Ad5 infection, P  = 0.0065 (**); for difference in recruitment of GFP-SMARCAL1-ΔRPA to VRCs relative to that of wt GFP-SMARCAL1 following Ad5 infection, P  = 8.8E−05 (****); for difference in recruitment of GFP-SMARCAL1-ΔP to VRCs relative to that of wt GFP-SMARCAL1 following Ad12 infection, P  = 0.04 (*); for difference in recruitment of GFP-SMARCAL1-ΔRPA to VRCs relative to that of wt GFP-SMARCAL1 following Ad5 infection, P  = 0.002 (***).
Figure Legend Snippet: SMARCAL1 is recruited to VRCs in an RPA-dependent and ATR- and CDK-dependent manner. (A) Microscopic images depicting the cellular localization of wt GFP-SMARCAL1, GFP-SMARCAL1-ΔP, and GFP-SMARCAL1-ΔRPA in mock-infected (i to iii), wt Ad5-infected (iv to vi), or wt Ad12-infected cells (vii to ix) 18 h postinfection. (B) Bar graph (± SEM) showing the percentage of GFP-labeled cells that are recruited to VRCs following Ad5 or Ad12 infection. n  = 3 (300 cells per experiment, 900 cells in total). Only those cells that exhibited clear GFP-SMARCAL1 structures in Ad-infected cells, comparable to the known architecture of VRCs at different stages of infection, were counted as VRC positive. Data presented were subjected to analysis of variance with a two-tailed t test. For significance testing for difference in recruitment of GFP-SMARCAL1-ΔP to VRCs relative to that of the wt GFP-SMARCAL1 following Ad5 infection, P  = 0.0065 (**); for difference in recruitment of GFP-SMARCAL1-ΔRPA to VRCs relative to that of wt GFP-SMARCAL1 following Ad5 infection, P  = 8.8E−05 (****); for difference in recruitment of GFP-SMARCAL1-ΔP to VRCs relative to that of wt GFP-SMARCAL1 following Ad12 infection, P  = 0.04 (*); for difference in recruitment of GFP-SMARCAL1-ΔRPA to VRCs relative to that of wt GFP-SMARCAL1 following Ad5 infection, P  = 0.002 (***).

Techniques Used: Recombinase Polymerase Amplification, Infection, Labeling, Two Tailed Test

SMARCAL1 is phosphorylated during the early stages of Ad infection. (A) A549 cells were either mock infected, treated with MLN4924, or infected with 10 PFU/cell of wt Ad5 or wt Ad12 and harvested at 18 h postinfection. Cells were harvested in IP buffer and subjected to immunoprecipitation for SMARCAL1. Anti-SMARCAL1 immunoprecipitates collected on protein G-Sepharose were treated in the absence or presence of λ-phosphatase and then subjected to SDS-PAGE and WB for SMARCAL1. (B) SMARCAL1 was immunoprecipitated from mock-infected and wt Ad5- or wt Ad12-infected A549 cells 18 h postinfection and separated by SDS-PAGE. Protein bands excised from the gel were subjected to trypsinization and mass spectrometric analysis. Identified SMARCAL1 phosphorylated peptides from Ad-infected cells are presented. (C) S123, S129, and S173 are conserved between primates but less well conserved in lower mammals. SMARCAL1 primary sequences from a number of species were aligned using CLUSTAL Omega. Shaded areas indicate conserved residues.
Figure Legend Snippet: SMARCAL1 is phosphorylated during the early stages of Ad infection. (A) A549 cells were either mock infected, treated with MLN4924, or infected with 10 PFU/cell of wt Ad5 or wt Ad12 and harvested at 18 h postinfection. Cells were harvested in IP buffer and subjected to immunoprecipitation for SMARCAL1. Anti-SMARCAL1 immunoprecipitates collected on protein G-Sepharose were treated in the absence or presence of λ-phosphatase and then subjected to SDS-PAGE and WB for SMARCAL1. (B) SMARCAL1 was immunoprecipitated from mock-infected and wt Ad5- or wt Ad12-infected A549 cells 18 h postinfection and separated by SDS-PAGE. Protein bands excised from the gel were subjected to trypsinization and mass spectrometric analysis. Identified SMARCAL1 phosphorylated peptides from Ad-infected cells are presented. (C) S123, S129, and S173 are conserved between primates but less well conserved in lower mammals. SMARCAL1 primary sequences from a number of species were aligned using CLUSTAL Omega. Shaded areas indicate conserved residues.

Techniques Used: Infection, Immunoprecipitation, SDS Page, Western Blot

SMARCAL1 is reorganized to viral replication centers during the early stages of Ad infection. A549 cells were either mock infected (i to iii) or infected with 10 PFU/cell of wt Ad5 (iv to vi) or wt Ad12 (vii to ix). At 18 h postinfection, cells were fixed, permeabilized, and costained for SMARCAL1 and RPA2. Arrows indicate regions of RPA2/SMARCAL1 colocalization. In all instances, images were recorded using a Zeiss LSM510-Meta confocal microscope.
Figure Legend Snippet: SMARCAL1 is reorganized to viral replication centers during the early stages of Ad infection. A549 cells were either mock infected (i to iii) or infected with 10 PFU/cell of wt Ad5 (iv to vi) or wt Ad12 (vii to ix). At 18 h postinfection, cells were fixed, permeabilized, and costained for SMARCAL1 and RPA2. Arrows indicate regions of RPA2/SMARCAL1 colocalization. In all instances, images were recorded using a Zeiss LSM510-Meta confocal microscope.

Techniques Used: Infection, Microscopy

Ad E1B-55K associates with SMARCAL1 in Ad-transformed cells. (A) Ad E1B-55K and SMARCAL1 were immunoprecipitated from Ad5 HEK 293 cells (A) and Ad12 HER2 cells (B) and subjected to WB for E1B-55K and SMARCAL1. IgG, immunoglobulin control IP.
Figure Legend Snippet: Ad E1B-55K associates with SMARCAL1 in Ad-transformed cells. (A) Ad E1B-55K and SMARCAL1 were immunoprecipitated from Ad5 HEK 293 cells (A) and Ad12 HER2 cells (B) and subjected to WB for E1B-55K and SMARCAL1. IgG, immunoglobulin control IP.

Techniques Used: Transformation Assay, Immunoprecipitation, Western Blot

ATR kinase and CDKs promote SMARCAL1 degradation following Ad5 and Ad12 infection. A549 cells were either mock infected or infected with 10 PFU/cell of wt Ad5 (A and C) or wt Ad12 (B and D). Cells were then incubated in the absence or presence of ATR inhibitor (AZD6738 [ATRi], 1 μM; A and B) or ATR and CDK inhibitors (AZD6738, 1 μM and RO-3306 [CDKi], 9 μM; C and D) and harvested at the appropriate times postinfection. Cell lysates were then separated by SDS-PAGE and subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of three independent experiments.
Figure Legend Snippet: ATR kinase and CDKs promote SMARCAL1 degradation following Ad5 and Ad12 infection. A549 cells were either mock infected or infected with 10 PFU/cell of wt Ad5 (A and C) or wt Ad12 (B and D). Cells were then incubated in the absence or presence of ATR inhibitor (AZD6738 [ATRi], 1 μM; A and B) or ATR and CDK inhibitors (AZD6738, 1 μM and RO-3306 [CDKi], 9 μM; C and D) and harvested at the appropriate times postinfection. Cell lysates were then separated by SDS-PAGE and subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of three independent experiments.

Techniques Used: Infection, Incubation, SDS Page, Western Blot

18) Product Images from "A green fluorescent protein-reporter mammalian two-hybrid system with extrachromosomal maintenance of a prey expression plasmid: Application to interaction screening"

Article Title: A green fluorescent protein-reporter mammalian two-hybrid system with extrachromosomal maintenance of a prey expression plasmid: Application to interaction screening

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

doi:

Diagram of the GFP-reporter mammalian two-hybrid system with extrachromosomal maintenance of prey expression plasmid. GB133 cells, which express the EBNA-1 protein, harbor a GAL4-dependent GFP reporter plasmid in their chromosomes. For interaction screening, GB133 cells can be stably transfected with an expression plasmid for a GAL4 DNA-binding domain (GAL4DB) fusion protein of bait. Cells can then be transiently transfected with a cDNA library expressing fusion proteins of a transcriptional activating domain (AD) and preys. The two-hybrid interaction will induce transcription of the GFP gene, which is readily detectable by fluorescent microscopy of living cells. Once introduced into GB133 cells by transient transfection protocols, the prey-expressing plasmids harboring the OriP replication origin sequence will be maintained stably because of the presence of the EBNA-1 protein, thus keeping the positive prey-expressing cells permanently green fluorescent. GAL4BE, GAL4 binding element; TATA , TATAA box from adenovirus E1b gene promoter.
Figure Legend Snippet: Diagram of the GFP-reporter mammalian two-hybrid system with extrachromosomal maintenance of prey expression plasmid. GB133 cells, which express the EBNA-1 protein, harbor a GAL4-dependent GFP reporter plasmid in their chromosomes. For interaction screening, GB133 cells can be stably transfected with an expression plasmid for a GAL4 DNA-binding domain (GAL4DB) fusion protein of bait. Cells can then be transiently transfected with a cDNA library expressing fusion proteins of a transcriptional activating domain (AD) and preys. The two-hybrid interaction will induce transcription of the GFP gene, which is readily detectable by fluorescent microscopy of living cells. Once introduced into GB133 cells by transient transfection protocols, the prey-expressing plasmids harboring the OriP replication origin sequence will be maintained stably because of the presence of the EBNA-1 protein, thus keeping the positive prey-expressing cells permanently green fluorescent. GAL4BE, GAL4 binding element; TATA , TATAA box from adenovirus E1b gene promoter.

Techniques Used: Expressing, Plasmid Preparation, Stable Transfection, Transfection, Binding Assay, cDNA Library Assay, Microscopy, Sequencing

19) Product Images from "Differential Roles of Smad1 and p38 Kinase in Regulation of Peroxisome Proliferator-activating Receptor ? during Bone Morphogenetic Protein 2-induced Adipogenesis"

Article Title: Differential Roles of Smad1 and p38 Kinase in Regulation of Peroxisome Proliferator-activating Receptor ? during Bone Morphogenetic Protein 2-induced Adipogenesis

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E02-06-0356

Schematic model of BMP2-induced adipogenesis. Activation of Smad1 is essential for induction of PPARγ expression, which is critical for adipogenic effects of BMP2. In contrast, activation of p38 kinase, presumably through MKK3 and MKK6, plays roles in the up-regulation of transcriptional activity of PPARγ.
Figure Legend Snippet: Schematic model of BMP2-induced adipogenesis. Activation of Smad1 is essential for induction of PPARγ expression, which is critical for adipogenic effects of BMP2. In contrast, activation of p38 kinase, presumably through MKK3 and MKK6, plays roles in the up-regulation of transcriptional activity of PPARγ.

Techniques Used: Activation Assay, Expressing, Activity Assay

(A) Overexpression of TAK1- and TAB1-induced activation of p38 kinase in C3H10T1/2 cells. C3H10T1/2 cells were transfected with pcDNA3 vector or both TAK1 and TAB1 expression constructs. Twenty hours after transfection, the cells were serum starved for 16 h and lysed. The cell lysates were determined by immunoblotting with anti-phospho-p38 kinase (top) or anti-p38 kinase (bottom) antibodies. (B) Overexpression of TAK1 and TAB1 up-regulated transcriptional activity of PPARγ. pcDNA3 (control) (1.6 μg) or both TAK1 (0.8 μg) and TAB1 (0.8 μg) expression vectors (1.6 μg) were transfected into C3H10T1/2 cells together with PPARγ (0.1 μg) and RXR (0.1 μg) expression vectors and PPRE-Luc (0.1 μg) and TK-renilla (0.1 μg) reporter constructs. Twelve hours after transfection, cells were incubated with or without SB203580 (10 μM) for 2 d. At the end of the culture, cells were lysed and the luciferase activity was measured and normalized by determining renilla luciferase activity as described in the text. Data were shown as mean ± SD. (C) Dominant-negative MKK3 inhibited BMP2-induced adipogenesis in C3H10T1/2 cells. C3H10T1/2 cells infected with control or dominant-negative MKK3 adenovirus at 100 multiplicity of infection were incubated with or without BMP2 (300 ng/ml). The cells were stained with Oil Red O, photographed under a microscope, and the areas stained with Oil Red O in the cells were assessed. (D) SB203580 inhibited BMP2-induced adipogenesis. C3H10T1/2 cells were incubated with BMP2 (300 ng/ml), SB203580 (10 μM), or both for 10 d. The cells were stained with Oil Red O, photographed under a microscope, and the areas stained with Oil Red O in the cells were assessed.
Figure Legend Snippet: (A) Overexpression of TAK1- and TAB1-induced activation of p38 kinase in C3H10T1/2 cells. C3H10T1/2 cells were transfected with pcDNA3 vector or both TAK1 and TAB1 expression constructs. Twenty hours after transfection, the cells were serum starved for 16 h and lysed. The cell lysates were determined by immunoblotting with anti-phospho-p38 kinase (top) or anti-p38 kinase (bottom) antibodies. (B) Overexpression of TAK1 and TAB1 up-regulated transcriptional activity of PPARγ. pcDNA3 (control) (1.6 μg) or both TAK1 (0.8 μg) and TAB1 (0.8 μg) expression vectors (1.6 μg) were transfected into C3H10T1/2 cells together with PPARγ (0.1 μg) and RXR (0.1 μg) expression vectors and PPRE-Luc (0.1 μg) and TK-renilla (0.1 μg) reporter constructs. Twelve hours after transfection, cells were incubated with or without SB203580 (10 μM) for 2 d. At the end of the culture, cells were lysed and the luciferase activity was measured and normalized by determining renilla luciferase activity as described in the text. Data were shown as mean ± SD. (C) Dominant-negative MKK3 inhibited BMP2-induced adipogenesis in C3H10T1/2 cells. C3H10T1/2 cells infected with control or dominant-negative MKK3 adenovirus at 100 multiplicity of infection were incubated with or without BMP2 (300 ng/ml). The cells were stained with Oil Red O, photographed under a microscope, and the areas stained with Oil Red O in the cells were assessed. (D) SB203580 inhibited BMP2-induced adipogenesis. C3H10T1/2 cells were incubated with BMP2 (300 ng/ml), SB203580 (10 μM), or both for 10 d. The cells were stained with Oil Red O, photographed under a microscope, and the areas stained with Oil Red O in the cells were assessed.

Techniques Used: Over Expression, Activation Assay, Transfection, Plasmid Preparation, Expressing, Construct, Activity Assay, Incubation, Luciferase, Dominant Negative Mutation, Infection, Staining, Microscopy

20) Product Images from "The Human Cytomegalovirus Major Immediate-Early Distal Enhancer Region Is Required for Efficient Viral Replication and Immediate-Early Gene Expression"

Article Title: The Human Cytomegalovirus Major Immediate-Early Distal Enhancer Region Is Required for Efficient Viral Replication and Immediate-Early Gene Expression

Journal: Journal of Virology

doi:

Schematic diagram of recombinant HCMVs containing mutations of the MIE regulatory region. HCMV genome and its unique long (U L ) and short (U S ), internal repeat long (IR L ) and short (IR S ), terminal repeat long (TR L ) and short (TR S ), and a-sequence components are depicted. Locations of MIE regulatory region, MIE gene exons 1 to 4 (open boxes), and putative UL128 gene (open box) within U L component are shown. The MIE regulatory region of the WT is composed of proximal promoter (+1 to −64), enhancer (Enh; -65 to -550), unique region (-551 to -749), and modulator (Mod; -750 to -1140). Numerical base positions are assigned relative to start site of MIE RNAs. Recombinant HCMVs rΔ-300/-640 SVgfp , rΔ-300/-1108 SVgfp , rΔ-640/-1108 SVgfp , and r SVgfp were derived from rΔM SVgpt ). rΔ-300/-640 SVgfp , rΔ-300/-1108 SVgfp , and rΔ-640/-1108 SVgfp have deletions from -300 to -640, -300 to -1108, and -640 to -1108, respectively. An enhancerless SV40 early promoter (−138 to +57), gfp ORF, and SV40 early intron and polyadenylation signal were inserted at the site of deletion. r SVgfp has the same insertion at -640 but has no deletion. rΔM SVgpt has a deletion of -640 to -1108 and insertion of the SV40 early transcription unit containing the gpt ORF.
Figure Legend Snippet: Schematic diagram of recombinant HCMVs containing mutations of the MIE regulatory region. HCMV genome and its unique long (U L ) and short (U S ), internal repeat long (IR L ) and short (IR S ), terminal repeat long (TR L ) and short (TR S ), and a-sequence components are depicted. Locations of MIE regulatory region, MIE gene exons 1 to 4 (open boxes), and putative UL128 gene (open box) within U L component are shown. The MIE regulatory region of the WT is composed of proximal promoter (+1 to −64), enhancer (Enh; -65 to -550), unique region (-551 to -749), and modulator (Mod; -750 to -1140). Numerical base positions are assigned relative to start site of MIE RNAs. Recombinant HCMVs rΔ-300/-640 SVgfp , rΔ-300/-1108 SVgfp , rΔ-640/-1108 SVgfp , and r SVgfp were derived from rΔM SVgpt ). rΔ-300/-640 SVgfp , rΔ-300/-1108 SVgfp , and rΔ-640/-1108 SVgfp have deletions from -300 to -640, -300 to -1108, and -640 to -1108, respectively. An enhancerless SV40 early promoter (−138 to +57), gfp ORF, and SV40 early intron and polyadenylation signal were inserted at the site of deletion. r SVgfp has the same insertion at -640 but has no deletion. rΔM SVgpt has a deletion of -640 to -1108 and insertion of the SV40 early transcription unit containing the gpt ORF.

Techniques Used: Recombinant, Sequencing, Derivative Assay

21) Product Images from "Multiple Signal Input and Output Domains of the 160-Kilodalton Nuclear Receptor Coactivator Proteins"

Article Title: Multiple Signal Input and Output Domains of the 160-Kilodalton Nuclear Receptor Coactivator Proteins

Journal: Molecular and Cellular Biology

doi:

NR box-independent and AD1-independent enhancement of AR AF-1 activity by GRIP1 and its C-terminal fragment. CV-1 cells were cotransfected with 0.25 μg of pM vector encoding Gal4DBD-ARAF1, 0.5 μg of GK1 reporter gene, and the indicated amount of pSG5.HA vector encoding GRIP1 or GRIP1 fragments. Luciferase activity of the cell extracts is shown.
Figure Legend Snippet: NR box-independent and AD1-independent enhancement of AR AF-1 activity by GRIP1 and its C-terminal fragment. CV-1 cells were cotransfected with 0.25 μg of pM vector encoding Gal4DBD-ARAF1, 0.5 μg of GK1 reporter gene, and the indicated amount of pSG5.HA vector encoding GRIP1 or GRIP1 fragments. Luciferase activity of the cell extracts is shown.

Techniques Used: Activity Assay, Plasmid Preparation, Luciferase

22) Product Images from "Microphthalmia-associated transcription factor interacts with LEF-1, a mediator of Wnt signaling"

Article Title: Microphthalmia-associated transcription factor interacts with LEF-1, a mediator of Wnt signaling

Journal: The EMBO Journal

doi: 10.1093/emboj/21.11.2703

Fig. 8. Functional cooperation of LEF-1 with MITF-related proteins. ( A ) Comparison of the bHLH/LZ regions. ( B ) Cooperation of LEF-1 with various bHLH/LZ proteins. The amino acid identity is shown as a percentage of the MITF bHLH/LZ region. HeLa cells were cotransfected with pHDTL8 and the indicated combination of LEF-1 and various bHLH/LZ proteins. An equal amount of MITF constructs was used (2 µg each), and the total amounts of plasmid DNA were maintained at 4 µg with the vector DNA (pRc/CMV). The data are presented as the ratio of normalized luciferase activity obtained with each combination and that obtained with vector DNA.
Figure Legend Snippet: Fig. 8. Functional cooperation of LEF-1 with MITF-related proteins. ( A ) Comparison of the bHLH/LZ regions. ( B ) Cooperation of LEF-1 with various bHLH/LZ proteins. The amino acid identity is shown as a percentage of the MITF bHLH/LZ region. HeLa cells were cotransfected with pHDTL8 and the indicated combination of LEF-1 and various bHLH/LZ proteins. An equal amount of MITF constructs was used (2 µg each), and the total amounts of plasmid DNA were maintained at 4 µg with the vector DNA (pRc/CMV). The data are presented as the ratio of normalized luciferase activity obtained with each combination and that obtained with vector DNA.

Techniques Used: Functional Assay, Construct, Plasmid Preparation, Luciferase, Activity Assay

Fig. 7. Identification of a crucial amino acid in the bHLH/LZ region of MITF-M for protein interaction. ( A ) The amino acid substitutions in the bHLH/LZ region (asterisks). ( B ) Effects on functional cooperation. HeLa cells were cotransfected with a DCT-luciferase reporter plasmid (pHDTL8), LEF-1 and the indicated MITF-M proteins. The results of three independent experiments are shown with standard deviations. ( C ) Effects on physical interaction. Two-hybrid assays were performed in 293 human embryonic kidney cells using the fusion proteins containing a bHLH/LZ region of MITF-M or its mutant proteins.
Figure Legend Snippet: Fig. 7. Identification of a crucial amino acid in the bHLH/LZ region of MITF-M for protein interaction. ( A ) The amino acid substitutions in the bHLH/LZ region (asterisks). ( B ) Effects on functional cooperation. HeLa cells were cotransfected with a DCT-luciferase reporter plasmid (pHDTL8), LEF-1 and the indicated MITF-M proteins. The results of three independent experiments are shown with standard deviations. ( C ) Effects on physical interaction. Two-hybrid assays were performed in 293 human embryonic kidney cells using the fusion proteins containing a bHLH/LZ region of MITF-M or its mutant proteins.

Techniques Used: Functional Assay, Luciferase, Plasmid Preparation, Mutagenesis

Fig. 6. Interaction of the bHLH/LZ region of MITF-M with LEF-1. ( A ) In vitro interaction between LEF-1 and the bHLH/LZ domain of MITF. COS-7 nuclear extracts contained endogenous c-Myc (lanes 1–12), as indicated by an arrow. Tagged LEF-1 was detected as enhanced signals in the fractions, bound to truncated MITF-M proteins containing the bHLH/LZ domain (lanes 9–11). An arrowhead indicates the unspecific protein binding (lanes 1–7). ( B ) Interaction between LEF-1 and the bHLH/LZ region of MITF-M in yeast cells.
Figure Legend Snippet: Fig. 6. Interaction of the bHLH/LZ region of MITF-M with LEF-1. ( A ) In vitro interaction between LEF-1 and the bHLH/LZ domain of MITF. COS-7 nuclear extracts contained endogenous c-Myc (lanes 1–12), as indicated by an arrow. Tagged LEF-1 was detected as enhanced signals in the fractions, bound to truncated MITF-M proteins containing the bHLH/LZ domain (lanes 9–11). An arrowhead indicates the unspecific protein binding (lanes 1–7). ( B ) Interaction between LEF-1 and the bHLH/LZ region of MITF-M in yeast cells.

Techniques Used: In Vitro, Protein Binding

23) Product Images from "A green fluorescent protein-reporter mammalian two-hybrid system with extrachromosomal maintenance of a prey expression plasmid: Application to interaction screening"

Article Title: A green fluorescent protein-reporter mammalian two-hybrid system with extrachromosomal maintenance of a prey expression plasmid: Application to interaction screening

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

doi:

GFP expression in GB133 cells. ( A ) Two-hybrid dependent expression of GFP in GB133 cells. Cells were transiently transfected with expression plasmids for a model bait (GAL4DB-fusion Smad4) and prey (MSG1) together with a β-galactosidase (β-Gal) reporter plasmid. Expression of GFP or β-galactosidase was evaluated 48 h after transfection. Wt + AD, wild-type prey with the transactivating domain; WtΔAD, wild-type prey lacking the transactivating domain; Mut + AD, prey mutant with the transactivating domain that does not interact with the bait. ( B ) GB133 cells maintain OriP -containing plasmids. Cells were transfected transiently with expression plasmids for GAL4DB or a GAL4DB-fusion CR2 transactivating domain (AD) with or without harboring the OriP sequence. GFP expression was evaluated 7 days after transfection, when cells formed confluent monolayers (phase contrast images are not shown).
Figure Legend Snippet: GFP expression in GB133 cells. ( A ) Two-hybrid dependent expression of GFP in GB133 cells. Cells were transiently transfected with expression plasmids for a model bait (GAL4DB-fusion Smad4) and prey (MSG1) together with a β-galactosidase (β-Gal) reporter plasmid. Expression of GFP or β-galactosidase was evaluated 48 h after transfection. Wt + AD, wild-type prey with the transactivating domain; WtΔAD, wild-type prey lacking the transactivating domain; Mut + AD, prey mutant with the transactivating domain that does not interact with the bait. ( B ) GB133 cells maintain OriP -containing plasmids. Cells were transfected transiently with expression plasmids for GAL4DB or a GAL4DB-fusion CR2 transactivating domain (AD) with or without harboring the OriP sequence. GFP expression was evaluated 7 days after transfection, when cells formed confluent monolayers (phase contrast images are not shown).

Techniques Used: Expressing, Transfection, Plasmid Preparation, Mutagenesis, Sequencing

Diagram of the GFP-reporter mammalian two-hybrid system with extrachromosomal maintenance of prey expression plasmid. GB133 cells, which express the EBNA-1 protein, harbor a GAL4-dependent GFP reporter plasmid in their chromosomes. For interaction screening, GB133 cells can be stably transfected with an expression plasmid for a GAL4 DNA-binding domain (GAL4DB) fusion protein of bait. Cells can then be transiently transfected with a cDNA library expressing fusion proteins of a transcriptional activating domain (AD) and preys. The two-hybrid interaction will induce transcription of the GFP gene, which is readily detectable by fluorescent microscopy of living cells. Once introduced into GB133 cells by transient transfection protocols, the prey-expressing plasmids harboring the OriP replication origin sequence will be maintained stably because of the presence of the EBNA-1 protein, thus keeping the positive prey-expressing cells permanently green fluorescent. GAL4BE, GAL4 binding element; TATA , TATAA box from adenovirus E1b gene promoter.
Figure Legend Snippet: Diagram of the GFP-reporter mammalian two-hybrid system with extrachromosomal maintenance of prey expression plasmid. GB133 cells, which express the EBNA-1 protein, harbor a GAL4-dependent GFP reporter plasmid in their chromosomes. For interaction screening, GB133 cells can be stably transfected with an expression plasmid for a GAL4 DNA-binding domain (GAL4DB) fusion protein of bait. Cells can then be transiently transfected with a cDNA library expressing fusion proteins of a transcriptional activating domain (AD) and preys. The two-hybrid interaction will induce transcription of the GFP gene, which is readily detectable by fluorescent microscopy of living cells. Once introduced into GB133 cells by transient transfection protocols, the prey-expressing plasmids harboring the OriP replication origin sequence will be maintained stably because of the presence of the EBNA-1 protein, thus keeping the positive prey-expressing cells permanently green fluorescent. GAL4BE, GAL4 binding element; TATA , TATAA box from adenovirus E1b gene promoter.

Techniques Used: Expressing, Plasmid Preparation, Stable Transfection, Transfection, Binding Assay, cDNA Library Assay, Microscopy, Sequencing

Recovery of a model prey (MSG1) plasmid from GB133 cells stably expressing a model bait (GAL4DB-Smad4). ( A–C ) Cells were transiently transfected with a prey ( B and C ) or empty vector ( A ), followed by evaluation of GFP expression by fluorescence microscopy at 48 h after transfection (fluorescent light only in A and B ; fluorescent light plus weak white light in C ). Arrows indicate strongly green fluorescent cells; arrowhead indicates a weakly green fluorescent cell. ( D–I ) Maintenance of OriP -harboring plasmid in bait-expressing GB133 cells. Expression plasmids for a GAL4DB-fusion transactivating domain with ( E ) or without ( D and F ) harboring the OriP replication origin sequence were transiently transfected into the bait-expressing GB133 cells, and expression of GFP was evaluated at 48 h ( D ) or at 7 days ( E and F ) after transfection. ( G–I ) Phase contrast images of cell monolayers corresponding to fluorescence images of D–F are shown in panels G–I , respectively. ( J and K ) Detection of prey diluted with an excess of empty vectors. Cells were transiently transfected with an OriP -containing prey expression plasmid together with a 2,000-fold molar excess of empty vector, followed by evaluation of GFP expression at 7 days after transfection. Single prey-transfected cells divided three times after transfection, forming green fluorescent cell clusters, each of which consisted of six to eight GFP-expressing cells (fluorescent light only in J ; fluorescent light plus weak white light in K ). ( L ) A green fluorescent microcolony of prey-transfected cells 3 days after subculture of the GFP-expressing cell clusters shown in J (fluorescent light plus weak white light). ( M ) Recovery of prey cDNA from green fluorescent microcolonies by PCR using primers that annealed to the vector sequences flanking the insert-cloning site. Agarose gel electrophoresis of PCR products is shown. Lane 1 , positive control amplification from the prey plasmid; lane 2, negative control amplification from empty vector; lanes 3–8, amplification of prey cDNA from total DNA preparations of five independent GFP-expressing microcolonies.
Figure Legend Snippet: Recovery of a model prey (MSG1) plasmid from GB133 cells stably expressing a model bait (GAL4DB-Smad4). ( A–C ) Cells were transiently transfected with a prey ( B and C ) or empty vector ( A ), followed by evaluation of GFP expression by fluorescence microscopy at 48 h after transfection (fluorescent light only in A and B ; fluorescent light plus weak white light in C ). Arrows indicate strongly green fluorescent cells; arrowhead indicates a weakly green fluorescent cell. ( D–I ) Maintenance of OriP -harboring plasmid in bait-expressing GB133 cells. Expression plasmids for a GAL4DB-fusion transactivating domain with ( E ) or without ( D and F ) harboring the OriP replication origin sequence were transiently transfected into the bait-expressing GB133 cells, and expression of GFP was evaluated at 48 h ( D ) or at 7 days ( E and F ) after transfection. ( G–I ) Phase contrast images of cell monolayers corresponding to fluorescence images of D–F are shown in panels G–I , respectively. ( J and K ) Detection of prey diluted with an excess of empty vectors. Cells were transiently transfected with an OriP -containing prey expression plasmid together with a 2,000-fold molar excess of empty vector, followed by evaluation of GFP expression at 7 days after transfection. Single prey-transfected cells divided three times after transfection, forming green fluorescent cell clusters, each of which consisted of six to eight GFP-expressing cells (fluorescent light only in J ; fluorescent light plus weak white light in K ). ( L ) A green fluorescent microcolony of prey-transfected cells 3 days after subculture of the GFP-expressing cell clusters shown in J (fluorescent light plus weak white light). ( M ) Recovery of prey cDNA from green fluorescent microcolonies by PCR using primers that annealed to the vector sequences flanking the insert-cloning site. Agarose gel electrophoresis of PCR products is shown. Lane 1 , positive control amplification from the prey plasmid; lane 2, negative control amplification from empty vector; lanes 3–8, amplification of prey cDNA from total DNA preparations of five independent GFP-expressing microcolonies.

Techniques Used: Plasmid Preparation, Stable Transfection, Expressing, Transfection, Fluorescence, Microscopy, Sequencing, Polymerase Chain Reaction, Clone Assay, Agarose Gel Electrophoresis, Positive Control, Amplification, Negative Control

24) 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

25) 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

26) Product Images from "A green fluorescent protein-reporter mammalian two-hybrid system with extrachromosomal maintenance of a prey expression plasmid: Application to interaction screening"

Article Title: A green fluorescent protein-reporter mammalian two-hybrid system with extrachromosomal maintenance of a prey expression plasmid: Application to interaction screening

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

doi:

Diagram of the GFP-reporter mammalian two-hybrid system with extrachromosomal maintenance of prey expression plasmid. GB133 cells, which express the EBNA-1 protein, harbor a GAL4-dependent GFP reporter plasmid in their chromosomes. For interaction screening, GB133 cells can be stably transfected with an expression plasmid for a GAL4 DNA-binding domain (GAL4DB) fusion protein of bait. Cells can then be transiently transfected with a cDNA library expressing fusion proteins of a transcriptional activating domain (AD) and preys. The two-hybrid interaction will induce transcription of the GFP gene, which is readily detectable by fluorescent microscopy of living cells. Once introduced into GB133 cells by transient transfection protocols, the prey-expressing plasmids harboring the OriP replication origin sequence will be maintained stably because of the presence of the EBNA-1 protein, thus keeping the positive prey-expressing cells permanently green fluorescent. GAL4BE, GAL4 binding element; TATA , TATAA box from adenovirus E1b gene promoter.
Figure Legend Snippet: Diagram of the GFP-reporter mammalian two-hybrid system with extrachromosomal maintenance of prey expression plasmid. GB133 cells, which express the EBNA-1 protein, harbor a GAL4-dependent GFP reporter plasmid in their chromosomes. For interaction screening, GB133 cells can be stably transfected with an expression plasmid for a GAL4 DNA-binding domain (GAL4DB) fusion protein of bait. Cells can then be transiently transfected with a cDNA library expressing fusion proteins of a transcriptional activating domain (AD) and preys. The two-hybrid interaction will induce transcription of the GFP gene, which is readily detectable by fluorescent microscopy of living cells. Once introduced into GB133 cells by transient transfection protocols, the prey-expressing plasmids harboring the OriP replication origin sequence will be maintained stably because of the presence of the EBNA-1 protein, thus keeping the positive prey-expressing cells permanently green fluorescent. GAL4BE, GAL4 binding element; TATA , TATAA box from adenovirus E1b gene promoter.

Techniques Used: Expressing, Plasmid Preparation, Stable Transfection, Transfection, Binding Assay, cDNA Library Assay, Microscopy, Sequencing

27) Product Images from "Adenovirus E1B 55-Kilodalton Protein Targets SMARCAL1 for Degradation during Infection and Modulates Cellular DNA Replication"

Article Title: Adenovirus E1B 55-Kilodalton Protein Targets SMARCAL1 for Degradation during Infection and Modulates Cellular DNA Replication

Journal: Journal of Virology

doi: 10.1128/JVI.00402-19

SMARCAL1 is recruited to VRCs in an RPA-dependent and ATR- and CDK-dependent manner. (A) Microscopic images depicting the cellular localization of wt GFP-SMARCAL1, GFP-SMARCAL1-ΔP, and GFP-SMARCAL1-ΔRPA in mock-infected (i to iii), wt Ad5-infected (iv to vi), or wt Ad12-infected cells (vii to ix) 18 h postinfection. (B) Bar graph (± SEM) showing the percentage of GFP-labeled cells that are recruited to VRCs following Ad5 or Ad12 infection. n = 3 (300 cells per experiment, 900 cells in total). Only those cells that exhibited clear GFP-SMARCAL1 structures in Ad-infected cells, comparable to the known architecture of VRCs at different stages of infection, were counted as VRC positive. Data presented were subjected to analysis of variance with a two-tailed t test. For significance testing for difference in recruitment of GFP-SMARCAL1-ΔP to VRCs relative to that of the wt GFP-SMARCAL1 following Ad5 infection, P = 0.0065 (**); for difference in recruitment of GFP-SMARCAL1-ΔRPA to VRCs relative to that of wt GFP-SMARCAL1 following Ad5 infection, P = 8.8E−05 (****); for difference in recruitment of GFP-SMARCAL1-ΔP to VRCs relative to that of wt GFP-SMARCAL1 following Ad12 infection, P = 0.04 (*); for difference in recruitment of GFP-SMARCAL1-ΔRPA to VRCs relative to that of wt GFP-SMARCAL1 following Ad5 infection, P = 0.002 (***).
Figure Legend Snippet: SMARCAL1 is recruited to VRCs in an RPA-dependent and ATR- and CDK-dependent manner. (A) Microscopic images depicting the cellular localization of wt GFP-SMARCAL1, GFP-SMARCAL1-ΔP, and GFP-SMARCAL1-ΔRPA in mock-infected (i to iii), wt Ad5-infected (iv to vi), or wt Ad12-infected cells (vii to ix) 18 h postinfection. (B) Bar graph (± SEM) showing the percentage of GFP-labeled cells that are recruited to VRCs following Ad5 or Ad12 infection. n = 3 (300 cells per experiment, 900 cells in total). Only those cells that exhibited clear GFP-SMARCAL1 structures in Ad-infected cells, comparable to the known architecture of VRCs at different stages of infection, were counted as VRC positive. Data presented were subjected to analysis of variance with a two-tailed t test. For significance testing for difference in recruitment of GFP-SMARCAL1-ΔP to VRCs relative to that of the wt GFP-SMARCAL1 following Ad5 infection, P = 0.0065 (**); for difference in recruitment of GFP-SMARCAL1-ΔRPA to VRCs relative to that of wt GFP-SMARCAL1 following Ad5 infection, P = 8.8E−05 (****); for difference in recruitment of GFP-SMARCAL1-ΔP to VRCs relative to that of wt GFP-SMARCAL1 following Ad12 infection, P = 0.04 (*); for difference in recruitment of GFP-SMARCAL1-ΔRPA to VRCs relative to that of wt GFP-SMARCAL1 following Ad5 infection, P = 0.002 (***).

Techniques Used: Recombinase Polymerase Amplification, Infection, Labeling, Two Tailed Test

28) Product Images from "A green fluorescent protein-reporter mammalian two-hybrid system with extrachromosomal maintenance of a prey expression plasmid: Application to interaction screening"

Article Title: A green fluorescent protein-reporter mammalian two-hybrid system with extrachromosomal maintenance of a prey expression plasmid: Application to interaction screening

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

doi:

Diagram of the GFP-reporter mammalian two-hybrid system with extrachromosomal maintenance of prey expression plasmid. GB133 cells, which express the EBNA-1 protein, harbor a GAL4-dependent GFP reporter plasmid in their chromosomes. For interaction screening, GB133 cells can be stably transfected with an expression plasmid for a GAL4 DNA-binding domain (GAL4DB) fusion protein of bait. Cells can then be transiently transfected with a cDNA library expressing fusion proteins of a transcriptional activating domain (AD) and preys. The two-hybrid interaction will induce transcription of the GFP gene, which is readily detectable by fluorescent microscopy of living cells. Once introduced into GB133 cells by transient transfection protocols, the prey-expressing plasmids harboring the OriP replication origin sequence will be maintained stably because of the presence of the EBNA-1 protein, thus keeping the positive prey-expressing cells permanently green fluorescent. GAL4BE, GAL4 binding element; TATA , TATAA box from adenovirus E1b gene promoter.
Figure Legend Snippet: Diagram of the GFP-reporter mammalian two-hybrid system with extrachromosomal maintenance of prey expression plasmid. GB133 cells, which express the EBNA-1 protein, harbor a GAL4-dependent GFP reporter plasmid in their chromosomes. For interaction screening, GB133 cells can be stably transfected with an expression plasmid for a GAL4 DNA-binding domain (GAL4DB) fusion protein of bait. Cells can then be transiently transfected with a cDNA library expressing fusion proteins of a transcriptional activating domain (AD) and preys. The two-hybrid interaction will induce transcription of the GFP gene, which is readily detectable by fluorescent microscopy of living cells. Once introduced into GB133 cells by transient transfection protocols, the prey-expressing plasmids harboring the OriP replication origin sequence will be maintained stably because of the presence of the EBNA-1 protein, thus keeping the positive prey-expressing cells permanently green fluorescent. GAL4BE, GAL4 binding element; TATA , TATAA box from adenovirus E1b gene promoter.

Techniques Used: Expressing, Plasmid Preparation, Stable Transfection, Transfection, Binding Assay, cDNA Library Assay, Microscopy, Sequencing

29) Product Images from "Paired-Homeodomain Transcription Factor PAX4 Acts as a Transcriptional Repressor in Early Pancreatic Development"

Article Title: Paired-Homeodomain Transcription Factor PAX4 Acts as a Transcriptional Repressor in Early Pancreatic Development

Journal: Molecular and Cellular Biology

doi:

PAX4 repressor domains. (A) Comparison of PAX4 and PAX6 structure. PD, paired domain; HD, homeodomain. (B) Transient transfections of the indicated cell lines were performed with the chimeric PAX4-GAL4 DBD fusion protein constructs shown and a reporter construct consisting of the GAL4 binding site linked to HSV-TK driving CAT expression (GAL4UAS-TK-CAT). (C) Transient transfections of βTC3 and NIH3T3 cells were performed with a reporter construct consisting of five copies of the GAL4 GAL4 UAS. (D) Transient transfections of αTC1.6 cells were performed with a reporter construct consisting of HSV-TK driving CAT expression without a GAL4 binding site (TK-CAT). (E) Transient transfections of βTC3 cells were performed with a reporter consisting of five GAL4 binding sites linked to the minimal adenovirus E1b promoter driving CAT expression (5XGAL4UAS-E1b-CAT). Note that the lowermost expression construct consists of the PAX6 C-terminal domain fused to the GAL4 DBD. Transfections were performed in duplicate on at least three separate occasions. Relative CAT activity of the GAL4-DBD expression construct alone is set arbitrarily at +1. Graphs show mean ± standard error of the mean.
Figure Legend Snippet: PAX4 repressor domains. (A) Comparison of PAX4 and PAX6 structure. PD, paired domain; HD, homeodomain. (B) Transient transfections of the indicated cell lines were performed with the chimeric PAX4-GAL4 DBD fusion protein constructs shown and a reporter construct consisting of the GAL4 binding site linked to HSV-TK driving CAT expression (GAL4UAS-TK-CAT). (C) Transient transfections of βTC3 and NIH3T3 cells were performed with a reporter construct consisting of five copies of the GAL4 GAL4 UAS. (D) Transient transfections of αTC1.6 cells were performed with a reporter construct consisting of HSV-TK driving CAT expression without a GAL4 binding site (TK-CAT). (E) Transient transfections of βTC3 cells were performed with a reporter consisting of five GAL4 binding sites linked to the minimal adenovirus E1b promoter driving CAT expression (5XGAL4UAS-E1b-CAT). Note that the lowermost expression construct consists of the PAX6 C-terminal domain fused to the GAL4 DBD. Transfections were performed in duplicate on at least three separate occasions. Relative CAT activity of the GAL4-DBD expression construct alone is set arbitrarily at +1. Graphs show mean ± standard error of the mean.

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

30) Product Images from "Paired-Homeodomain Transcription Factor PAX4 Acts as a Transcriptional Repressor in Early Pancreatic Development"

Article Title: Paired-Homeodomain Transcription Factor PAX4 Acts as a Transcriptional Repressor in Early Pancreatic Development

Journal: Molecular and Cellular Biology

doi:

PAX4 transcriptional repression in fetal pancreatic epithelium. Transient transfections were performed in mouse e11.5 pancreatic epithelium with expression vectors containing the GAL4 DBD coding sequence fused with the PAX4 and PAX6 cDNA fragments shown, the pFOXLuc2.TK.5GAL reporter plasmid, and the pFOXEGFP.CMV internal standard. Transfections were performed in triplicate on individual fetal pancreatic buds. Luciferase mRNA levels were measured by RT-PCR and standardized to EGFP mRNA levels. The graph shows mean ± standard error of the mean.
Figure Legend Snippet: PAX4 transcriptional repression in fetal pancreatic epithelium. Transient transfections were performed in mouse e11.5 pancreatic epithelium with expression vectors containing the GAL4 DBD coding sequence fused with the PAX4 and PAX6 cDNA fragments shown, the pFOXLuc2.TK.5GAL reporter plasmid, and the pFOXEGFP.CMV internal standard. Transfections were performed in triplicate on individual fetal pancreatic buds. Luciferase mRNA levels were measured by RT-PCR and standardized to EGFP mRNA levels. The graph shows mean ± standard error of the mean.

Techniques Used: Transfection, Expressing, Sequencing, Plasmid Preparation, Luciferase, Reverse Transcription Polymerase Chain Reaction

PAX4 repressor domains. (A) Comparison of PAX4 and PAX6 structure. PD, paired domain; HD, homeodomain. (B) Transient transfections of the indicated cell lines were performed with the chimeric PAX4-GAL4 DBD fusion protein constructs shown and a reporter construct consisting of the GAL4 binding site linked to HSV-TK driving CAT expression (GAL4UAS-TK-CAT). (C) Transient transfections of βTC3 and NIH3T3 cells were performed with a reporter construct consisting of five copies of the GAL4 GAL4 UAS. (D) Transient transfections of αTC1.6 cells were performed with a reporter construct consisting of HSV-TK driving CAT expression without a GAL4 binding site (TK-CAT). (E) Transient transfections of βTC3 cells were performed with a reporter consisting of five GAL4 binding sites linked to the minimal adenovirus E1b promoter driving CAT expression (5XGAL4UAS-E1b-CAT). Note that the lowermost expression construct consists of the PAX6 C-terminal domain fused to the GAL4 DBD. Transfections were performed in duplicate on at least three separate occasions. Relative CAT activity of the GAL4-DBD expression construct alone is set arbitrarily at +1. Graphs show mean ± standard error of the mean.
Figure Legend Snippet: PAX4 repressor domains. (A) Comparison of PAX4 and PAX6 structure. PD, paired domain; HD, homeodomain. (B) Transient transfections of the indicated cell lines were performed with the chimeric PAX4-GAL4 DBD fusion protein constructs shown and a reporter construct consisting of the GAL4 binding site linked to HSV-TK driving CAT expression (GAL4UAS-TK-CAT). (C) Transient transfections of βTC3 and NIH3T3 cells were performed with a reporter construct consisting of five copies of the GAL4 GAL4 UAS. (D) Transient transfections of αTC1.6 cells were performed with a reporter construct consisting of HSV-TK driving CAT expression without a GAL4 binding site (TK-CAT). (E) Transient transfections of βTC3 cells were performed with a reporter consisting of five GAL4 binding sites linked to the minimal adenovirus E1b promoter driving CAT expression (5XGAL4UAS-E1b-CAT). Note that the lowermost expression construct consists of the PAX6 C-terminal domain fused to the GAL4 DBD. Transfections were performed in duplicate on at least three separate occasions. Relative CAT activity of the GAL4-DBD expression construct alone is set arbitrarily at +1. Graphs show mean ± standard error of the mean.

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

31) Product Images from "Differential Roles of Smad1 and p38 Kinase in Regulation of Peroxisome Proliferator-activating Receptor ? during Bone Morphogenetic Protein 2-induced Adipogenesis"

Article Title: Differential Roles of Smad1 and p38 Kinase in Regulation of Peroxisome Proliferator-activating Receptor ? during Bone Morphogenetic Protein 2-induced Adipogenesis

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E02-06-0356

Overexpression of Smad6 inhibited activation of Smad1 (A), BMP2-regulated induction of PPARγ (B), and adipogenesis (C and D). (A) C3H10T1/2 cells infected with control or Flag-Smad6 adenovirus at 100 multiplicity of infection were starved with 0.2% fetal calf serum-DMEM for 16 h and then stimulated with or without BMP2 (300 ng/ml) for 10 min and lysed. The cell lysates were immunprecipitated with anti-Smad1 antibody and immunoblotted with anti-phsophoserine antibody (top). The amount of Smad1 in the immunoprecipitates was determined by immunoblotting with anti-Smad1 (second panel). The lysates were also determined by immunoblotting with antiphsopho-p38 (third panel) or antip38 (forth panel) antibodies. The expression of Flag-Smad6 was determined by immunoblotting with anti-Flag antibody (bottom). (B) C3H10T1/2 cells infected with control or Flag-Smad6 adenovirus at 100 multiplicity of infection were incubated with or without BMP2 (300 ng/ml) for 4 d and lysed. The lysates were determined by immunoblotting with anti-PPARγ mAb followed by immunoprecipitation with anti-PPARγ polyclonal antibody (top). The lysates were also determined by immunoblotting with anti-Flag (middle) or anti-β-actin antibodies (bottom). (C and D) 10T1/2 cells infected with control or Flag-Smad6 adenovirus at 100 multiplicity of infection were incubated with or without BMP2 (300 ng/ml) for 10 d. The cells were stained with Oil Red O, photographed under a microscope, and the areas stained with Oil Red O in the cells were assessed.
Figure Legend Snippet: Overexpression of Smad6 inhibited activation of Smad1 (A), BMP2-regulated induction of PPARγ (B), and adipogenesis (C and D). (A) C3H10T1/2 cells infected with control or Flag-Smad6 adenovirus at 100 multiplicity of infection were starved with 0.2% fetal calf serum-DMEM for 16 h and then stimulated with or without BMP2 (300 ng/ml) for 10 min and lysed. The cell lysates were immunprecipitated with anti-Smad1 antibody and immunoblotted with anti-phsophoserine antibody (top). The amount of Smad1 in the immunoprecipitates was determined by immunoblotting with anti-Smad1 (second panel). The lysates were also determined by immunoblotting with antiphsopho-p38 (third panel) or antip38 (forth panel) antibodies. The expression of Flag-Smad6 was determined by immunoblotting with anti-Flag antibody (bottom). (B) C3H10T1/2 cells infected with control or Flag-Smad6 adenovirus at 100 multiplicity of infection were incubated with or without BMP2 (300 ng/ml) for 4 d and lysed. The lysates were determined by immunoblotting with anti-PPARγ mAb followed by immunoprecipitation with anti-PPARγ polyclonal antibody (top). The lysates were also determined by immunoblotting with anti-Flag (middle) or anti-β-actin antibodies (bottom). (C and D) 10T1/2 cells infected with control or Flag-Smad6 adenovirus at 100 multiplicity of infection were incubated with or without BMP2 (300 ng/ml) for 10 d. The cells were stained with Oil Red O, photographed under a microscope, and the areas stained with Oil Red O in the cells were assessed.

Techniques Used: Over Expression, Activation Assay, Infection, Expressing, Incubation, Immunoprecipitation, Staining, Microscopy

(A) BMP2 up-regulated the transcriptional activity of PPARγ in C3H10T1/2 cells. Left, pcDNA3 (control) (1.6 μg) or Smad6 expression vector (1.6 μg) was transfected into C3H10T1/2 cells together with PPARγ (0.1 μg) and RXR (0.1 μg) expression vectors and PPRE-Luc (0.1 μg) and TK-renilla (0.1 μg) reporter constructs. Twelve hours after transfection, cells were incubated with or without BMP2 (300 ng/ml) for 2 d. At the end of the culture, cells were lysed and the luciferase activity was measured and normalized by determining renilla luciferase activity as described in the text. Data were shown as mean ± SD. Right, expression of PPARγ and Smad6 were monitored by immunoblotting with anti-PPARγ (top) and anti-Flag (middle) antibodies in the same set of transfection experiments. The lysates were also determined by immunoblotting with anti-β-actin antibody (bottom). (B) Introduction of PPARγ rescued the adipogenesis blocked by Smad6. C3H10T1/2 cells were infected with Flag-Smad6 (100 multiplicity of infection) and/or PPARγ (100 multiplicity of infection) adenoviruses and incubated with or without BMP2 (300 ng/ml) for 7 d. The cells were stained with Oil Red O, photographed under a microscope, and the areas stained with Oil Red O in the cells were assessed.
Figure Legend Snippet: (A) BMP2 up-regulated the transcriptional activity of PPARγ in C3H10T1/2 cells. Left, pcDNA3 (control) (1.6 μg) or Smad6 expression vector (1.6 μg) was transfected into C3H10T1/2 cells together with PPARγ (0.1 μg) and RXR (0.1 μg) expression vectors and PPRE-Luc (0.1 μg) and TK-renilla (0.1 μg) reporter constructs. Twelve hours after transfection, cells were incubated with or without BMP2 (300 ng/ml) for 2 d. At the end of the culture, cells were lysed and the luciferase activity was measured and normalized by determining renilla luciferase activity as described in the text. Data were shown as mean ± SD. Right, expression of PPARγ and Smad6 were monitored by immunoblotting with anti-PPARγ (top) and anti-Flag (middle) antibodies in the same set of transfection experiments. The lysates were also determined by immunoblotting with anti-β-actin antibody (bottom). (B) Introduction of PPARγ rescued the adipogenesis blocked by Smad6. C3H10T1/2 cells were infected with Flag-Smad6 (100 multiplicity of infection) and/or PPARγ (100 multiplicity of infection) adenoviruses and incubated with or without BMP2 (300 ng/ml) for 7 d. The cells were stained with Oil Red O, photographed under a microscope, and the areas stained with Oil Red O in the cells were assessed.

Techniques Used: Activity Assay, Expressing, Plasmid Preparation, Transfection, Construct, Incubation, Luciferase, Infection, Staining, Microscopy

32) Product Images from "RhoA Interacts with the Fusion Glycoprotein of Respiratory Syncytial Virus and Facilitates Virus-Induced Syncytium Formation"

Article Title: RhoA Interacts with the Fusion Glycoprotein of Respiratory Syncytial Virus and Facilitates Virus-Induced Syncytium Formation

Journal: Journal of Virology

doi:

F-RhoA interaction analysis in the mammalian two-hybrid system. The level of CAT enzyme activity detected in a culture of HEp-2 cells transfected with pM and pG5CAT reporter plasmid was set at 1.0. Levels of CAT expression observed when HEp-2 cultures were additionally cotransfected with the indicated VP16 fusion protein expression plasmids are given as multiples of this value. Cotransfection with pM3-VP16 (encoding a fusion of the Gal4 DNA-BD and the VP16 AD) and pG5CAT is the positive control.
Figure Legend Snippet: F-RhoA interaction analysis in the mammalian two-hybrid system. The level of CAT enzyme activity detected in a culture of HEp-2 cells transfected with pM and pG5CAT reporter plasmid was set at 1.0. Levels of CAT expression observed when HEp-2 cultures were additionally cotransfected with the indicated VP16 fusion protein expression plasmids are given as multiples of this value. Cotransfection with pM3-VP16 (encoding a fusion of the Gal4 DNA-BD and the VP16 AD) and pG5CAT is the positive control.

Techniques Used: Activity Assay, Transfection, Plasmid Preparation, Expressing, Cotransfection, Positive Control

33) Product Images from "Paired-Homeodomain Transcription Factor PAX4 Acts as a Transcriptional Repressor in Early Pancreatic Development"

Article Title: Paired-Homeodomain Transcription Factor PAX4 Acts as a Transcriptional Repressor in Early Pancreatic Development

Journal: Molecular and Cellular Biology

doi:

PAX4 transcriptional repression. Transient transfections of the indicated cell lines were performed with the PAX4 expression vector and reporter constructs consisting of the HSV-TK promoter alone (TK) or the isolated rInsIC2 element linked to TK-HSV (C2-TK) (A) and the −410-bp rat insulin I promoter (RIP), −482-bp rat glucagon promoter (GLU), RSV LTR (RSV), or CMV promoter (CMV) driving CAT expression. Transfections were performed in duplicate on at least three separate occasions. Relative CAT activity of the TK-CAT reporter in the absence of PAX4 expression is set arbitrarily at +1. In panel B, relative CAT activity of each construct in the absence of PAX4 expression is set arbitrarily at +1. Graphs show mean ± standard error of the mean.
Figure Legend Snippet: PAX4 transcriptional repression. Transient transfections of the indicated cell lines were performed with the PAX4 expression vector and reporter constructs consisting of the HSV-TK promoter alone (TK) or the isolated rInsIC2 element linked to TK-HSV (C2-TK) (A) and the −410-bp rat insulin I promoter (RIP), −482-bp rat glucagon promoter (GLU), RSV LTR (RSV), or CMV promoter (CMV) driving CAT expression. Transfections were performed in duplicate on at least three separate occasions. Relative CAT activity of the TK-CAT reporter in the absence of PAX4 expression is set arbitrarily at +1. In panel B, relative CAT activity of each construct in the absence of PAX4 expression is set arbitrarily at +1. Graphs show mean ± standard error of the mean.

Techniques Used: Transfection, Expressing, Plasmid Preparation, Construct, Isolation, Activity Assay

PAX4 repressor domains. (A) Comparison of PAX4 and PAX6 structure. PD, paired domain; HD, homeodomain. (B) Transient transfections of the indicated cell lines were performed with the chimeric PAX4-GAL4 DBD fusion protein constructs shown and a reporter construct consisting of the GAL4 binding site linked to HSV-TK driving CAT expression (GAL4UAS-TK-CAT). (C) Transient transfections of βTC3 and NIH3T3 cells were performed with a reporter construct consisting of five copies of the GAL4 GAL4 UAS. (D) Transient transfections of αTC1.6 cells were performed with a reporter construct consisting of HSV-TK driving CAT expression without a GAL4 binding site (TK-CAT). (E) Transient transfections of βTC3 cells were performed with a reporter consisting of five GAL4 binding sites linked to the minimal adenovirus E1b promoter driving CAT expression (5XGAL4UAS-E1b-CAT). Note that the lowermost expression construct consists of the PAX6 C-terminal domain fused to the GAL4 DBD. Transfections were performed in duplicate on at least three separate occasions. Relative CAT activity of the GAL4-DBD expression construct alone is set arbitrarily at +1. Graphs show mean ± standard error of the mean.
Figure Legend Snippet: PAX4 repressor domains. (A) Comparison of PAX4 and PAX6 structure. PD, paired domain; HD, homeodomain. (B) Transient transfections of the indicated cell lines were performed with the chimeric PAX4-GAL4 DBD fusion protein constructs shown and a reporter construct consisting of the GAL4 binding site linked to HSV-TK driving CAT expression (GAL4UAS-TK-CAT). (C) Transient transfections of βTC3 and NIH3T3 cells were performed with a reporter construct consisting of five copies of the GAL4 GAL4 UAS. (D) Transient transfections of αTC1.6 cells were performed with a reporter construct consisting of HSV-TK driving CAT expression without a GAL4 binding site (TK-CAT). (E) Transient transfections of βTC3 cells were performed with a reporter consisting of five GAL4 binding sites linked to the minimal adenovirus E1b promoter driving CAT expression (5XGAL4UAS-E1b-CAT). Note that the lowermost expression construct consists of the PAX6 C-terminal domain fused to the GAL4 DBD. Transfections were performed in duplicate on at least three separate occasions. Relative CAT activity of the GAL4-DBD expression construct alone is set arbitrarily at +1. Graphs show mean ± standard error of the mean.

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

34) 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

35) Product Images from "Microphthalmia-associated transcription factor interacts with LEF-1, a mediator of Wnt signaling"

Article Title: Microphthalmia-associated transcription factor interacts with LEF-1, a mediator of Wnt signaling

Journal: The EMBO Journal

doi: 10.1093/emboj/21.11.2703

Fig. 7. Identification of a crucial amino acid in the bHLH/LZ region of MITF-M for protein interaction. ( A ) The amino acid substitutions in the bHLH/LZ region (asterisks). ( B ) Effects on functional cooperation. HeLa cells were cotransfected with a DCT-luciferase reporter plasmid (pHDTL8), LEF-1 and the indicated MITF-M proteins. The results of three independent experiments are shown with standard deviations. ( C ) Effects on physical interaction. Two-hybrid assays were performed in 293 human embryonic kidney cells using the fusion proteins containing a bHLH/LZ region of MITF-M or its mutant proteins.
Figure Legend Snippet: Fig. 7. Identification of a crucial amino acid in the bHLH/LZ region of MITF-M for protein interaction. ( A ) The amino acid substitutions in the bHLH/LZ region (asterisks). ( B ) Effects on functional cooperation. HeLa cells were cotransfected with a DCT-luciferase reporter plasmid (pHDTL8), LEF-1 and the indicated MITF-M proteins. The results of three independent experiments are shown with standard deviations. ( C ) Effects on physical interaction. Two-hybrid assays were performed in 293 human embryonic kidney cells using the fusion proteins containing a bHLH/LZ region of MITF-M or its mutant proteins.

Techniques Used: Functional Assay, Luciferase, Plasmid Preparation, Mutagenesis

Fig. 1. Effects of LEF-1 on the DCT promoter activity. ( A ) Expression profiles of LEF-1 and TCF-1 mRNAs determined by RT–PCR. Note the faint band of GAPDH in mouse melan-a cells. ( B ) Promoter-context dependent transactivation of the DCT promoter by MITF-M and LEF-1. Each of the DCT reporter plasmids was coexpressed in HeLa cells with MITF-M, LEF-1 or a combination of MITF-M and LEF-1. An enhancer DDE1 (positions –447 to –416) and the M box (positions –138 to –128) are indicated. The magnitude of activation is presented as the ratio of normalized luciferase activity obtained with each plasmid and that with vector DNA (Induction Ratio). The results of at least three independent experiments are shown with standard deviations. ( C ) Activation of DCT promoter by LiCl. Melan-a cells, maintained in a 6-well plate, were transfected with the indicated constructs (1 µg each of reporter and effector and 0.05 µg of pCDNA3-His-LacZ), incubated for 20 h, and then treated with 30 mM LiCl for 24 h in fresh medium. The data are shown as a ratio to the basal luciferase activity obtained with pHDTL1. Other conditions were the same as in (B).
Figure Legend Snippet: Fig. 1. Effects of LEF-1 on the DCT promoter activity. ( A ) Expression profiles of LEF-1 and TCF-1 mRNAs determined by RT–PCR. Note the faint band of GAPDH in mouse melan-a cells. ( B ) Promoter-context dependent transactivation of the DCT promoter by MITF-M and LEF-1. Each of the DCT reporter plasmids was coexpressed in HeLa cells with MITF-M, LEF-1 or a combination of MITF-M and LEF-1. An enhancer DDE1 (positions –447 to –416) and the M box (positions –138 to –128) are indicated. The magnitude of activation is presented as the ratio of normalized luciferase activity obtained with each plasmid and that with vector DNA (Induction Ratio). The results of at least three independent experiments are shown with standard deviations. ( C ) Activation of DCT promoter by LiCl. Melan-a cells, maintained in a 6-well plate, were transfected with the indicated constructs (1 µg each of reporter and effector and 0.05 µg of pCDNA3-His-LacZ), incubated for 20 h, and then treated with 30 mM LiCl for 24 h in fresh medium. The data are shown as a ratio to the basal luciferase activity obtained with pHDTL1. Other conditions were the same as in (B).

Techniques Used: Activity Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Activation Assay, Luciferase, Plasmid Preparation, Transfection, Construct, Incubation

Fig. 2. Identification of the cis -acting element that is required for the activation of DCT promoter by LEF-1 and MITF-M. ( A ) Deletion studies of the DCT promoter. HeLa cells were cotransfected with the indicated reporter plasmids. ( B ) The cis -acting region in the DCT promoter. The 12-bp cis -acting element is underlined in DTPR2. Also shown is the strategy for the functional analysis in HeLa cells ( C ) and EMSAs ( D ). Base changes shown were introduced into construct pHDTL12. Nuclear extracts of HeLa cells were incubated with a 32 P-end labeled DTPR2 in the absence (lanes 2, 3, 14 and 15) or presence of an indicated competitor (200- and 500-fold excesses, shown as triangles). LEFBS represents a LEF-1-binding site. Lanes 1 and 13 represent a control lacking nuclear extracts. Arrows 1 and 2 indicate the specific and unspecific protein–DNA complexes, respectively. Unbound probes are indicated by arrow 3.
Figure Legend Snippet: Fig. 2. Identification of the cis -acting element that is required for the activation of DCT promoter by LEF-1 and MITF-M. ( A ) Deletion studies of the DCT promoter. HeLa cells were cotransfected with the indicated reporter plasmids. ( B ) The cis -acting region in the DCT promoter. The 12-bp cis -acting element is underlined in DTPR2. Also shown is the strategy for the functional analysis in HeLa cells ( C ) and EMSAs ( D ). Base changes shown were introduced into construct pHDTL12. Nuclear extracts of HeLa cells were incubated with a 32 P-end labeled DTPR2 in the absence (lanes 2, 3, 14 and 15) or presence of an indicated competitor (200- and 500-fold excesses, shown as triangles). LEFBS represents a LEF-1-binding site. Lanes 1 and 13 represent a control lacking nuclear extracts. Arrows 1 and 2 indicate the specific and unspecific protein–DNA complexes, respectively. Unbound probes are indicated by arrow 3.

Techniques Used: Activation Assay, Functional Assay, Construct, Incubation, Labeling, Binding Assay

Fig. 6. Interaction of the bHLH/LZ region of MITF-M with LEF-1. ( A ) In vitro interaction between LEF-1 and the bHLH/LZ domain of MITF. COS-7 nuclear extracts contained endogenous c-Myc (lanes 1–12), as indicated by an arrow. Tagged LEF-1 was detected as enhanced signals in the fractions, bound to truncated MITF-M proteins containing the bHLH/LZ domain (lanes 9–11). An arrowhead indicates the unspecific protein binding (lanes 1–7). ( B ) Interaction between LEF-1 and the bHLH/LZ region of MITF-M in yeast cells.
Figure Legend Snippet: Fig. 6. Interaction of the bHLH/LZ region of MITF-M with LEF-1. ( A ) In vitro interaction between LEF-1 and the bHLH/LZ domain of MITF. COS-7 nuclear extracts contained endogenous c-Myc (lanes 1–12), as indicated by an arrow. Tagged LEF-1 was detected as enhanced signals in the fractions, bound to truncated MITF-M proteins containing the bHLH/LZ domain (lanes 9–11). An arrowhead indicates the unspecific protein binding (lanes 1–7). ( B ) Interaction between LEF-1 and the bHLH/LZ region of MITF-M in yeast cells.

Techniques Used: In Vitro, Protein Binding

Fig. 3. Functional cooperation of MITF-M with LEF-1. ( A ) EMSAs showing the LEF-1-binding site of the DCT promoter. GST–LEF-1 fusion protein was incubated with a 32 ). An arrow indicates the specific protein–DNA complex. ( B ) Domains of LEF-1 required for the DCT promoter activation. HeLa cells were cotransfected with a DCT reporter plasmid, pHDTL8, and indicated LEF-1 plasmids. Shown are the N-terminal β-catenin-binding domain (β) and the NLS near the C-terminus (closed box). Reporter luciferase activity obtained was normalized with each β-galactosidase activity that represents an internal control. The magnitude of activation is presented as the ratio of normalized luciferase activity obtained with each effector plasmid and that with vector DNA. ( C ) Subcellular localization of EGFP–LEF-1 fusion proteins in COS-7 cells, as assessed by fluorescence.
Figure Legend Snippet: Fig. 3. Functional cooperation of MITF-M with LEF-1. ( A ) EMSAs showing the LEF-1-binding site of the DCT promoter. GST–LEF-1 fusion protein was incubated with a 32 ). An arrow indicates the specific protein–DNA complex. ( B ) Domains of LEF-1 required for the DCT promoter activation. HeLa cells were cotransfected with a DCT reporter plasmid, pHDTL8, and indicated LEF-1 plasmids. Shown are the N-terminal β-catenin-binding domain (β) and the NLS near the C-terminus (closed box). Reporter luciferase activity obtained was normalized with each β-galactosidase activity that represents an internal control. The magnitude of activation is presented as the ratio of normalized luciferase activity obtained with each effector plasmid and that with vector DNA. ( C ) Subcellular localization of EGFP–LEF-1 fusion proteins in COS-7 cells, as assessed by fluorescence.

Techniques Used: Functional Assay, Binding Assay, Incubation, Activation Assay, Plasmid Preparation, Luciferase, Activity Assay, Fluorescence

36) Product Images from "Triple-controlled oncolytic adenovirus expressing melittin to exert inhibitory efficacy on hepatocellular carcinoma"

Article Title: Triple-controlled oncolytic adenovirus expressing melittin to exert inhibitory efficacy on hepatocellular carcinoma

Journal: International Journal of Clinical and Experimental Pathology

doi:

A. Schematic representation of the adenovirus, in which in which the hybrid promoter HRE-AFP was used to regulate the E1a, and the E1b-55 kDa gene was deleted, the Melittin expressing cassette was inserted into E3 region. The negative control virus QG511-HA
Figure Legend Snippet: A. Schematic representation of the adenovirus, in which in which the hybrid promoter HRE-AFP was used to regulate the E1a, and the E1b-55 kDa gene was deleted, the Melittin expressing cassette was inserted into E3 region. The negative control virus QG511-HA

Techniques Used: Expressing, Negative Control

37) Product Images from "A CCAAT/Enhancer Binding Protein ? Isoform, Liver-Enriched Inhibitory Protein, Regulates Commitment of Osteoblasts and Adipocytes"

Article Title: A CCAAT/Enhancer Binding Protein ? Isoform, Liver-Enriched Inhibitory Protein, Regulates Commitment of Osteoblasts and Adipocytes

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.25.5.1971-1979.2005

C/EBPβ promotes osteoblast differentiation of mesenchymal cells. (A through C) C3H10T1/2 cells were infected with control or C/EBPβ adenovirus at an MOI of 40 and incubated with BMP2 for 7 days. The cells were determined by immunoblotting (A), ALP staining (B), or ALP activity (C). (D) C3H10T1/2 cells were infected with control, Runx2, or C/EBPβ adenovirus at an MOI of 40 and cultured for 7 days. Osteocalcin, type IA collagen (Col IA), or ALP expression in the cells was assessed by RT-PCR. (E) ST2, C2C12, and mouse primary mesenchymal cells were infected with control or C/EBPβ adenovirus at an MOI of 40 and incubated for 7 days. The cells were subjected to ALP staining.
Figure Legend Snippet: C/EBPβ promotes osteoblast differentiation of mesenchymal cells. (A through C) C3H10T1/2 cells were infected with control or C/EBPβ adenovirus at an MOI of 40 and incubated with BMP2 for 7 days. The cells were determined by immunoblotting (A), ALP staining (B), or ALP activity (C). (D) C3H10T1/2 cells were infected with control, Runx2, or C/EBPβ adenovirus at an MOI of 40 and cultured for 7 days. Osteocalcin, type IA collagen (Col IA), or ALP expression in the cells was assessed by RT-PCR. (E) ST2, C2C12, and mouse primary mesenchymal cells were infected with control or C/EBPβ adenovirus at an MOI of 40 and incubated for 7 days. The cells were subjected to ALP staining.

Techniques Used: Infection, Incubation, ALP Assay, Staining, Activity Assay, Cell Culture, IA, Expressing, Reverse Transcription Polymerase Chain Reaction

C/EBPβ expression associated with osteoblastogenesis. Expression of C/EBPβ and its isoform, LIP, in mouse primary osteoblasts as determined by immunoblotting analysis (A) and immunochytochemical analysis (B) is shown. C/EBPβ expression (C) and ALP activity (D) of C3H10T1/2 or primary mesenchymal cells cultured with or without BMP2 for 7 days are also shown.
Figure Legend Snippet: C/EBPβ expression associated with osteoblastogenesis. Expression of C/EBPβ and its isoform, LIP, in mouse primary osteoblasts as determined by immunoblotting analysis (A) and immunochytochemical analysis (B) is shown. C/EBPβ expression (C) and ALP activity (D) of C3H10T1/2 or primary mesenchymal cells cultured with or without BMP2 for 7 days are also shown.

Techniques Used: Expressing, ALP Assay, Activity Assay, Cell Culture

LIP inhibits adipocyte differentiation in a dominant-negative fashion. (A) Schematic structure of C/EBPβ, LIP, and its deletion mutants. AD, activation domain; BD, binding domain; LZ, leucine zipper. (B) C3H10T1/2 cells infected with control, C/EBPβ, or LIP adenovirus were analyzed by immunoblotting with anti-C/EBPβ antibody. (C) C3H10T1/2 cells were infected with adenoviruses as indicated in Materials and Methods, incubated for 7 days, and then stained with Oil red O. Oil red O-stained area was measured as described in Materials and Methods. (D) PPARγ gene promoter fused to the luciferase reporter construct and TK-renilla reporter constructs was transfected into C3H10T1/2 cells together with expression vectors as indicated. Luciferase activity of the cell lysates was measured. (E) C3H10T1/2 cells were incubated with BMP2 as indicated, and expression of LIP and C/EBPβ was determined by immunoblotting. (F) C3H10T1/2 cells infected with control or Flag-tagged LIP adenovirus were incubated with BMP2 for 7 days and stained with Oil red O. The Oil red O-stained area was measured. (G and H) C3H10T1/2 cells infected with control or Flag-tagged LIP adenovirus were incubated with BMP2 for 7 days and ALP activity (G) and osteocalcin production (H) were determined. (I) C3H10T1/2 cells infected with control or Flag-tagged LIP adenovirus were incubated with BMP2 for 7 days, and the cell lysates were examined by immunoblotting with anti-Flag, Runx2, or C/EBPβ antibody. (J) C2C12 cells infected with control or Flag-tagged LIP adenovirus were incubated with BMP2 for 7 days, and ALP activity was determined.
Figure Legend Snippet: LIP inhibits adipocyte differentiation in a dominant-negative fashion. (A) Schematic structure of C/EBPβ, LIP, and its deletion mutants. AD, activation domain; BD, binding domain; LZ, leucine zipper. (B) C3H10T1/2 cells infected with control, C/EBPβ, or LIP adenovirus were analyzed by immunoblotting with anti-C/EBPβ antibody. (C) C3H10T1/2 cells were infected with adenoviruses as indicated in Materials and Methods, incubated for 7 days, and then stained with Oil red O. Oil red O-stained area was measured as described in Materials and Methods. (D) PPARγ gene promoter fused to the luciferase reporter construct and TK-renilla reporter constructs was transfected into C3H10T1/2 cells together with expression vectors as indicated. Luciferase activity of the cell lysates was measured. (E) C3H10T1/2 cells were incubated with BMP2 as indicated, and expression of LIP and C/EBPβ was determined by immunoblotting. (F) C3H10T1/2 cells infected with control or Flag-tagged LIP adenovirus were incubated with BMP2 for 7 days and stained with Oil red O. The Oil red O-stained area was measured. (G and H) C3H10T1/2 cells infected with control or Flag-tagged LIP adenovirus were incubated with BMP2 for 7 days and ALP activity (G) and osteocalcin production (H) were determined. (I) C3H10T1/2 cells infected with control or Flag-tagged LIP adenovirus were incubated with BMP2 for 7 days, and the cell lysates were examined by immunoblotting with anti-Flag, Runx2, or C/EBPβ antibody. (J) C2C12 cells infected with control or Flag-tagged LIP adenovirus were incubated with BMP2 for 7 days, and ALP activity was determined.

Techniques Used: Dominant Negative Mutation, Activation Assay, Binding Assay, Infection, Incubation, Staining, Luciferase, Construct, Transfection, Expressing, Activity Assay, ALP Assay

C/EBPβ regulates osteoblastogenesis in a Runx2-dependent and -independent fashion. (A) C3H10T1/2 cells were infected with control or C/EBPβ adenovirus with or without Runx2 adenovirus and cultured for 7 days. ALP activity in the cells was measured. (B) Osteocalcin gene promoter fused to the luciferase reporter construct and TK-renilla reporter constructs were transfected into C3H10T1/2 cells together with pcDNA3 (control), the C/EBPβ expression vector, the Runx2 expression vector, or both vectors. Luciferase activity of the cell lysates was measured in relative light units (RLU). (C) The lysates of C3H10T1/2 cells infected with control or C/EBPβ adenovirus together with or without Runx2 adenovirus were examined by immunoblotting with anti-Runx2 or C/EBPβ antibody. (D) The lysates of C3H10T1/2 cells infected with control or C/EBPβ adenovirus were incubated with a biotinylated probe containing the C/EBP binding element (BE) in osteocalcin gene promoter in the presence or absence of unbiotinylated probe (Comp). Associated protein with biotinylated probe was determined by immunoblotting with anti-C/EBPβ antibody. Ppt, precipitation. (E) Luciferase reporter construct-fused osteocalcin gene promoter containing osteocalcin luciferase (OC-Luc) or lacking the C/EBP binding element [OC(ΔC/EBP-BE)-Luc] and the TK-renilla reporter construct were transfected into C3H10T1/2 cells together with pcDNA3 (control), the C/EBPβ expression vector, the Runx2 expression vector, or both vectors. Luciferase activity of the cell lysates was measured. (F) C3H10T1/2 cells were infected with the control, C/EBPβ adenovirus, Runx2 adenovirus, or both adenovirus strains. The cell lysates were immunoprecipitated (IP) with anti-C/EBPβ antibody, and immunoprecipitates were determined by immunoblotting with anti-Runx2 antibody. (G) C3T10T1/2 cells were infected with adenoviruses as indicated in the text and cultured for 7 days. ALP activity in the cells was measured. (H) Mesenchymal cells isolated from Runx2-deficient mice were infected with adenoviruses as indicated in the text and cultured for 7 days. ALP activity of the cells was measured.
Figure Legend Snippet: C/EBPβ regulates osteoblastogenesis in a Runx2-dependent and -independent fashion. (A) C3H10T1/2 cells were infected with control or C/EBPβ adenovirus with or without Runx2 adenovirus and cultured for 7 days. ALP activity in the cells was measured. (B) Osteocalcin gene promoter fused to the luciferase reporter construct and TK-renilla reporter constructs were transfected into C3H10T1/2 cells together with pcDNA3 (control), the C/EBPβ expression vector, the Runx2 expression vector, or both vectors. Luciferase activity of the cell lysates was measured in relative light units (RLU). (C) The lysates of C3H10T1/2 cells infected with control or C/EBPβ adenovirus together with or without Runx2 adenovirus were examined by immunoblotting with anti-Runx2 or C/EBPβ antibody. (D) The lysates of C3H10T1/2 cells infected with control or C/EBPβ adenovirus were incubated with a biotinylated probe containing the C/EBP binding element (BE) in osteocalcin gene promoter in the presence or absence of unbiotinylated probe (Comp). Associated protein with biotinylated probe was determined by immunoblotting with anti-C/EBPβ antibody. Ppt, precipitation. (E) Luciferase reporter construct-fused osteocalcin gene promoter containing osteocalcin luciferase (OC-Luc) or lacking the C/EBP binding element [OC(ΔC/EBP-BE)-Luc] and the TK-renilla reporter construct were transfected into C3H10T1/2 cells together with pcDNA3 (control), the C/EBPβ expression vector, the Runx2 expression vector, or both vectors. Luciferase activity of the cell lysates was measured. (F) C3H10T1/2 cells were infected with the control, C/EBPβ adenovirus, Runx2 adenovirus, or both adenovirus strains. The cell lysates were immunoprecipitated (IP) with anti-C/EBPβ antibody, and immunoprecipitates were determined by immunoblotting with anti-Runx2 antibody. (G) C3T10T1/2 cells were infected with adenoviruses as indicated in the text and cultured for 7 days. ALP activity in the cells was measured. (H) Mesenchymal cells isolated from Runx2-deficient mice were infected with adenoviruses as indicated in the text and cultured for 7 days. ALP activity of the cells was measured.

Techniques Used: Infection, Cell Culture, ALP Assay, Activity Assay, Luciferase, Construct, Transfection, Expressing, Plasmid Preparation, Incubation, Binding Assay, Immunoprecipitation, Isolation, Mouse Assay

LIP enhances osteogenic action of Runx2 through its physical association. (A) Total RNA of C3H10T1/2 cells infected with control (Cont) or Flag-LIP adenovirus or incubated with BMP2 was determined by RT-PCR analysis. (B) C3H10T1/2 cells were infected with adenoviruses as indicated in the text and incubated for 7 days. ALP activity of the cells was measured. (C) Osteocalcin gene promoters fused to luciferase reporter construct and TK-renilla reporter constructs were transfected into C3H10T1/2 cells together with pcDNA3 (control), LIP expression vector, Runx2 expression vector, or both vectors. Luciferase activity of the cell lysates was measured. (D) C2C12 cells infected with adenovirus as indicated in the text were cultured for 7 days and then examined by ALP staining. (E) C3H10T1/2 cells were infected with control, LIP, or Runx2 adenovirus or both LIP and Runx2 adenovirus. The cell lysates were immunoprecipitated with anti-C/EBPβ antibody, and immunoprecipitates (IP) were determined by immunoblotting with anti-Runx2 antibody. (F) The lysates of Cos7 cells were transfected with Myc-tagged LIP constructs as indicated and incubated with Flag-tagged Runx2 protein immobilized on protein G-agarose beads. Precipitated proteins (Ppt) were examined by immunoblotting with anti-Myc antibody. (G) The lysates of Cos7 cells were transfected with Flag-tagged Runx2 constructs as indicated and incubated with Myc-tagged LIP protein immobilized on protein G-agarose beads. Precipitated proteins were examined by immunoblotting with anti-Flag antibody. (H) The lysates of C3H10T1/2 cells infected with control or LIP adenovirus were incubated with biotinylated probe containing C/EBP binding element (BE) in the osteocalcin gene. Associated protein with biotinylated probe was determined by immunoblotting with anti-C/EBPβ antibody. (I) The lysates of C3H10T1/2 cells infected with control or Flag-tagged LIP adenovirus were subjected to chromatin immunoprecipitation analysis. C, immunoprecipitation with control mouse IgG; F, immunoprecipitation with anti-Flag antibody. (J) C3H10T1/2 cells were infected with adenoviruses as indicated in the text. Seven days after culture, ALP activity was examined. (K) Runx2-deficient mesenchymal cells were infected with control, either LIP adenovirus or Runx2 adenovirus, or both adenoviruses and then cultured for 7 days. ALP activity was determined.
Figure Legend Snippet: LIP enhances osteogenic action of Runx2 through its physical association. (A) Total RNA of C3H10T1/2 cells infected with control (Cont) or Flag-LIP adenovirus or incubated with BMP2 was determined by RT-PCR analysis. (B) C3H10T1/2 cells were infected with adenoviruses as indicated in the text and incubated for 7 days. ALP activity of the cells was measured. (C) Osteocalcin gene promoters fused to luciferase reporter construct and TK-renilla reporter constructs were transfected into C3H10T1/2 cells together with pcDNA3 (control), LIP expression vector, Runx2 expression vector, or both vectors. Luciferase activity of the cell lysates was measured. (D) C2C12 cells infected with adenovirus as indicated in the text were cultured for 7 days and then examined by ALP staining. (E) C3H10T1/2 cells were infected with control, LIP, or Runx2 adenovirus or both LIP and Runx2 adenovirus. The cell lysates were immunoprecipitated with anti-C/EBPβ antibody, and immunoprecipitates (IP) were determined by immunoblotting with anti-Runx2 antibody. (F) The lysates of Cos7 cells were transfected with Myc-tagged LIP constructs as indicated and incubated with Flag-tagged Runx2 protein immobilized on protein G-agarose beads. Precipitated proteins (Ppt) were examined by immunoblotting with anti-Myc antibody. (G) The lysates of Cos7 cells were transfected with Flag-tagged Runx2 constructs as indicated and incubated with Myc-tagged LIP protein immobilized on protein G-agarose beads. Precipitated proteins were examined by immunoblotting with anti-Flag antibody. (H) The lysates of C3H10T1/2 cells infected with control or LIP adenovirus were incubated with biotinylated probe containing C/EBP binding element (BE) in the osteocalcin gene. Associated protein with biotinylated probe was determined by immunoblotting with anti-C/EBPβ antibody. (I) The lysates of C3H10T1/2 cells infected with control or Flag-tagged LIP adenovirus were subjected to chromatin immunoprecipitation analysis. C, immunoprecipitation with control mouse IgG; F, immunoprecipitation with anti-Flag antibody. (J) C3H10T1/2 cells were infected with adenoviruses as indicated in the text. Seven days after culture, ALP activity was examined. (K) Runx2-deficient mesenchymal cells were infected with control, either LIP adenovirus or Runx2 adenovirus, or both adenoviruses and then cultured for 7 days. ALP activity was determined.

Techniques Used: Infection, Incubation, Reverse Transcription Polymerase Chain Reaction, ALP Assay, Activity Assay, Luciferase, Construct, Transfection, Expressing, Plasmid Preparation, Cell Culture, Staining, Immunoprecipitation, Binding Assay, Chromatin Immunoprecipitation

38) Product Images from "Modulation of Hepatic Granulomatous Responses by Transgene Expression of DAP12 or TREM-1-Ig Molecules"

Article Title: Modulation of Hepatic Granulomatous Responses by Transgene Expression of DAP12 or TREM-1-Ig Molecules

Journal: The American Journal of Pathology

doi:

A: Illustration of the mode by which Ad-FDAP12 and Ad-TREM-1 Ig vectors modulate the DAP12-mediated signaling pathway in myeloid lineage cells or monocytes/neutrophils. The membrane-anchored protein FDAP12 derived from Ad-FDAP12 is flagged in the diagram with circular shapes that distinguish from the endogenous DAP12 molecule. The signal through DAP12-associating molecules (TREM family, SIRPβ1, MDL-1) is transmitted to both FDAP12 and endogenous DAP12. TREM-1 Ig, the adenoviral-derived extracellular domain of TREM-1 that was linked to the human Ig Fc portion ( dotted in the diagram), serves as an inhibitor of DAP12 signaling pathway by competing with an as yet unidentified ligand for binding to the TREM-1 molecule. B: Construction of adenovirus vectors. The elements inserted into the adenoviral genome are illustrated in two bars ( top ) for Ad-FDAP12 and Ad-TREM-1 Ig vectors, respectively. The pAxCAwt cosmid vector containing the above insert was co-transfected into 293 cells with restriction enzyme-digested DNA-TPC (Ad genome tagged with 55-kd terminal protein) to generate recombinant adenoviruses. EC, Extracellular domain; CAG promoter, cytomegalovirus enhancer and chicken β-actin promoter; G poly A, rabbit β-globin poly A signal; FLAG, 24 nucleotides coding for eight defined amino acids (DYKDDDDK) serving as a tag; ApR, ampicillin-resistance gene; cos, cos site of λ phage; ori, replication origin.
Figure Legend Snippet: A: Illustration of the mode by which Ad-FDAP12 and Ad-TREM-1 Ig vectors modulate the DAP12-mediated signaling pathway in myeloid lineage cells or monocytes/neutrophils. The membrane-anchored protein FDAP12 derived from Ad-FDAP12 is flagged in the diagram with circular shapes that distinguish from the endogenous DAP12 molecule. The signal through DAP12-associating molecules (TREM family, SIRPβ1, MDL-1) is transmitted to both FDAP12 and endogenous DAP12. TREM-1 Ig, the adenoviral-derived extracellular domain of TREM-1 that was linked to the human Ig Fc portion ( dotted in the diagram), serves as an inhibitor of DAP12 signaling pathway by competing with an as yet unidentified ligand for binding to the TREM-1 molecule. B: Construction of adenovirus vectors. The elements inserted into the adenoviral genome are illustrated in two bars ( top ) for Ad-FDAP12 and Ad-TREM-1 Ig vectors, respectively. The pAxCAwt cosmid vector containing the above insert was co-transfected into 293 cells with restriction enzyme-digested DNA-TPC (Ad genome tagged with 55-kd terminal protein) to generate recombinant adenoviruses. EC, Extracellular domain; CAG promoter, cytomegalovirus enhancer and chicken β-actin promoter; G poly A, rabbit β-globin poly A signal; FLAG, 24 nucleotides coding for eight defined amino acids (DYKDDDDK) serving as a tag; ApR, ampicillin-resistance gene; cos, cos site of λ phage; ori, replication origin.

Techniques Used: Derivative Assay, Binding Assay, Plasmid Preparation, Transfection, Recombinant

Zymosan A-induced hepatic granuloma formation in Ad-DAP12-infected mice was augmented by anti-FLAG mAb. Twenty-four hours after zymosan A injection, mice were infected with Ad-FDAP12 plus isotype control mouse IgG1 ( A , D ), Ad-FDAP12 plus anti-FLAG mAb ( B , E ), and Ad-FDAP12 plus Ad-TREM-1 Ig plus anti-FLAG mAb ( C , F ). At day 7, mice were killed and liver sections were subjected to H E staining ( A–C ) and immunostaining for F4/80 ( D–F ). Original magnifications, ×100.
Figure Legend Snippet: Zymosan A-induced hepatic granuloma formation in Ad-DAP12-infected mice was augmented by anti-FLAG mAb. Twenty-four hours after zymosan A injection, mice were infected with Ad-FDAP12 plus isotype control mouse IgG1 ( A , D ), Ad-FDAP12 plus anti-FLAG mAb ( B , E ), and Ad-FDAP12 plus Ad-TREM-1 Ig plus anti-FLAG mAb ( C , F ). At day 7, mice were killed and liver sections were subjected to H E staining ( A–C ) and immunostaining for F4/80 ( D–F ). Original magnifications, ×100.

Techniques Used: Infection, Mouse Assay, Injection, Staining, Immunostaining

A: Gene expression of TREM-1 and FLAG-DAP12 in Ad-LacZ-, Ad-FDAP12-, and Ad-TREM-1 Ig-infected and zymosan A-injected mice. RT-PCR analysis was performed on total RNA extracted from mouse liver. Every liver of four mice in each group represents the same results. B: FLAG-DAP12 and endogenous DAP12 protein expression in F4/80-positive cells from Ad-FDAP12-infected mouse liver 3 days after zymosan A injection. F4/80-positive cells from four Ad-FDAP12-infected mice were collected. Lysates prepared from 1 × 10 6 cells were immunoprecipitated with anti-DAP12 polyclonal antibodies and analyzed by Western blotting using anti-DAP12 polyclonal antibodies or anti-FLAG mAb.
Figure Legend Snippet: A: Gene expression of TREM-1 and FLAG-DAP12 in Ad-LacZ-, Ad-FDAP12-, and Ad-TREM-1 Ig-infected and zymosan A-injected mice. RT-PCR analysis was performed on total RNA extracted from mouse liver. Every liver of four mice in each group represents the same results. B: FLAG-DAP12 and endogenous DAP12 protein expression in F4/80-positive cells from Ad-FDAP12-infected mouse liver 3 days after zymosan A injection. F4/80-positive cells from four Ad-FDAP12-infected mice were collected. Lysates prepared from 1 × 10 6 cells were immunoprecipitated with anti-DAP12 polyclonal antibodies and analyzed by Western blotting using anti-DAP12 polyclonal antibodies or anti-FLAG mAb.

Techniques Used: Expressing, Infection, Injection, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Western Blot

39) Product Images from "A green fluorescent protein-reporter mammalian two-hybrid system with extrachromosomal maintenance of a prey expression plasmid: Application to interaction screening"

Article Title: A green fluorescent protein-reporter mammalian two-hybrid system with extrachromosomal maintenance of a prey expression plasmid: Application to interaction screening

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

doi:

Diagram of the GFP-reporter mammalian two-hybrid system with extrachromosomal maintenance of prey expression plasmid. GB133 cells, which express the EBNA-1 protein, harbor a GAL4-dependent GFP reporter plasmid in their chromosomes. For interaction screening, GB133 cells can be stably transfected with an expression plasmid for a GAL4 DNA-binding domain (GAL4DB) fusion protein of bait. Cells can then be transiently transfected with a cDNA library expressing fusion proteins of a transcriptional activating domain (AD) and preys. The two-hybrid interaction will induce transcription of the GFP gene, which is readily detectable by fluorescent microscopy of living cells. Once introduced into GB133 cells by transient transfection protocols, the prey-expressing plasmids harboring the OriP replication origin sequence will be maintained stably because of the presence of the EBNA-1 protein, thus keeping the positive prey-expressing cells permanently green fluorescent. GAL4BE, GAL4 binding element; TATA , TATAA box from adenovirus E1b gene promoter.
Figure Legend Snippet: Diagram of the GFP-reporter mammalian two-hybrid system with extrachromosomal maintenance of prey expression plasmid. GB133 cells, which express the EBNA-1 protein, harbor a GAL4-dependent GFP reporter plasmid in their chromosomes. For interaction screening, GB133 cells can be stably transfected with an expression plasmid for a GAL4 DNA-binding domain (GAL4DB) fusion protein of bait. Cells can then be transiently transfected with a cDNA library expressing fusion proteins of a transcriptional activating domain (AD) and preys. The two-hybrid interaction will induce transcription of the GFP gene, which is readily detectable by fluorescent microscopy of living cells. Once introduced into GB133 cells by transient transfection protocols, the prey-expressing plasmids harboring the OriP replication origin sequence will be maintained stably because of the presence of the EBNA-1 protein, thus keeping the positive prey-expressing cells permanently green fluorescent. GAL4BE, GAL4 binding element; TATA , TATAA box from adenovirus E1b gene promoter.

Techniques Used: Expressing, Plasmid Preparation, Stable Transfection, Transfection, Binding Assay, cDNA Library Assay, Microscopy, Sequencing

Recovery of a model prey (MSG1) plasmid from GB133 cells stably expressing a model bait (GAL4DB-Smad4). ( A–C ) Cells were transiently transfected with a prey ( B and C ) or empty vector ( A ), followed by evaluation of GFP expression by fluorescence microscopy at 48 h after transfection (fluorescent light only in A and B ; fluorescent light plus weak white light in C ). Arrows indicate strongly green fluorescent cells; arrowhead indicates a weakly green fluorescent cell. ( D–I ) Maintenance of OriP -harboring plasmid in bait-expressing GB133 cells. Expression plasmids for a GAL4DB-fusion transactivating domain with ( E ) or without ( D and F ) harboring the OriP replication origin sequence were transiently transfected into the bait-expressing GB133 cells, and expression of GFP was evaluated at 48 h ( D ) or at 7 days ( E and F ) after transfection. ( G–I ) Phase contrast images of cell monolayers corresponding to fluorescence images of D–F are shown in panels G–I , respectively. ( J and K ) Detection of prey diluted with an excess of empty vectors. Cells were transiently transfected with an OriP -containing prey expression plasmid together with a 2,000-fold molar excess of empty vector, followed by evaluation of GFP expression at 7 days after transfection. Single prey-transfected cells divided three times after transfection, forming green fluorescent cell clusters, each of which consisted of six to eight GFP-expressing cells (fluorescent light only in J ; fluorescent light plus weak white light in K ). ( L ) A green fluorescent microcolony of prey-transfected cells 3 days after subculture of the GFP-expressing cell clusters shown in J (fluorescent light plus weak white light). ( M ) Recovery of prey cDNA from green fluorescent microcolonies by PCR using primers that annealed to the vector sequences flanking the insert-cloning site. Agarose gel electrophoresis of PCR products is shown. Lane 1 , positive control amplification from the prey plasmid; lane 2, negative control amplification from empty vector; lanes 3–8, amplification of prey cDNA from total DNA preparations of five independent GFP-expressing microcolonies.
Figure Legend Snippet: Recovery of a model prey (MSG1) plasmid from GB133 cells stably expressing a model bait (GAL4DB-Smad4). ( A–C ) Cells were transiently transfected with a prey ( B and C ) or empty vector ( A ), followed by evaluation of GFP expression by fluorescence microscopy at 48 h after transfection (fluorescent light only in A and B ; fluorescent light plus weak white light in C ). Arrows indicate strongly green fluorescent cells; arrowhead indicates a weakly green fluorescent cell. ( D–I ) Maintenance of OriP -harboring plasmid in bait-expressing GB133 cells. Expression plasmids for a GAL4DB-fusion transactivating domain with ( E ) or without ( D and F ) harboring the OriP replication origin sequence were transiently transfected into the bait-expressing GB133 cells, and expression of GFP was evaluated at 48 h ( D ) or at 7 days ( E and F ) after transfection. ( G–I ) Phase contrast images of cell monolayers corresponding to fluorescence images of D–F are shown in panels G–I , respectively. ( J and K ) Detection of prey diluted with an excess of empty vectors. Cells were transiently transfected with an OriP -containing prey expression plasmid together with a 2,000-fold molar excess of empty vector, followed by evaluation of GFP expression at 7 days after transfection. Single prey-transfected cells divided three times after transfection, forming green fluorescent cell clusters, each of which consisted of six to eight GFP-expressing cells (fluorescent light only in J ; fluorescent light plus weak white light in K ). ( L ) A green fluorescent microcolony of prey-transfected cells 3 days after subculture of the GFP-expressing cell clusters shown in J (fluorescent light plus weak white light). ( M ) Recovery of prey cDNA from green fluorescent microcolonies by PCR using primers that annealed to the vector sequences flanking the insert-cloning site. Agarose gel electrophoresis of PCR products is shown. Lane 1 , positive control amplification from the prey plasmid; lane 2, negative control amplification from empty vector; lanes 3–8, amplification of prey cDNA from total DNA preparations of five independent GFP-expressing microcolonies.

Techniques Used: Plasmid Preparation, Stable Transfection, Expressing, Transfection, Fluorescence, Microscopy, Sequencing, Polymerase Chain Reaction, Clone Assay, Agarose Gel Electrophoresis, Positive Control, Amplification, Negative Control

40) Product Images from "Paired-Homeodomain Transcription Factor PAX4 Acts as a Transcriptional Repressor in Early Pancreatic Development"

Article Title: Paired-Homeodomain Transcription Factor PAX4 Acts as a Transcriptional Repressor in Early Pancreatic Development

Journal: Molecular and Cellular Biology

doi:

PAX4 repressor domains. (A) Comparison of PAX4 and PAX6 structure. PD, paired domain; HD, homeodomain. (B) Transient transfections of the indicated cell lines were performed with the chimeric PAX4-GAL4 DBD fusion protein constructs shown and a reporter construct consisting of the GAL4 binding site linked to HSV-TK driving CAT expression (GAL4UAS-TK-CAT). (C) Transient transfections of βTC3 and NIH3T3 cells were performed with a reporter construct consisting of five copies of the GAL4 GAL4 UAS. (D) Transient transfections of αTC1.6 cells were performed with a reporter construct consisting of HSV-TK driving CAT expression without a GAL4 binding site (TK-CAT). (E) Transient transfections of βTC3 cells were performed with a reporter consisting of five GAL4 binding sites linked to the minimal adenovirus E1b promoter driving CAT expression (5XGAL4UAS-E1b-CAT). Note that the lowermost expression construct consists of the PAX6 C-terminal domain fused to the GAL4 DBD. Transfections were performed in duplicate on at least three separate occasions. Relative CAT activity of the GAL4-DBD expression construct alone is set arbitrarily at +1. Graphs show mean ± standard error of the mean.
Figure Legend Snippet: PAX4 repressor domains. (A) Comparison of PAX4 and PAX6 structure. PD, paired domain; HD, homeodomain. (B) Transient transfections of the indicated cell lines were performed with the chimeric PAX4-GAL4 DBD fusion protein constructs shown and a reporter construct consisting of the GAL4 binding site linked to HSV-TK driving CAT expression (GAL4UAS-TK-CAT). (C) Transient transfections of βTC3 and NIH3T3 cells were performed with a reporter construct consisting of five copies of the GAL4 GAL4 UAS. (D) Transient transfections of αTC1.6 cells were performed with a reporter construct consisting of HSV-TK driving CAT expression without a GAL4 binding site (TK-CAT). (E) Transient transfections of βTC3 cells were performed with a reporter consisting of five GAL4 binding sites linked to the minimal adenovirus E1b promoter driving CAT expression (5XGAL4UAS-E1b-CAT). Note that the lowermost expression construct consists of the PAX6 C-terminal domain fused to the GAL4 DBD. Transfections were performed in duplicate on at least three separate occasions. Relative CAT activity of the GAL4-DBD expression construct alone is set arbitrarily at +1. Graphs show mean ± standard error of the mean.

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

Related Articles

Clone Assay:

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RNA Extraction:

Article Title: Phosphoribosyl Pyrophosphate Amidotransferase Promotes the Progression of Thyroid Cancer via Regulating Pyruvate Kinase M2
Article Snippet: .. RNA Extraction and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) According to the manufacturer’s instructions, total RNA was extracted from TC tissues and cells using TRIzol (Invitrogen, Shanghai, China), and cDNA was prepared using the FastQuan RT kit (Tiangen Biotech., Beijing, China). qRT-PCR analysis was performed using SYBR Premix Ex Taq ™ Kit (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer’s instructions. .. Quantitative data were normalized to β-actin and the relative expression was calculated using the 2−ΔΔCt method.

Mutagenesis:

Article Title: Adenovirus E1B 55-Kilodalton Protein Targets SMARCAL1 for Degradation during Infection and Modulates Cellular DNA Replication
Article Snippet: .. wt SMARCAL1 and ΔN-SMARCAL1 (lacking the N-terminal RPA interaction domain; ΔRPA) constructs cloned into the retroviral vector pLEGFP-C1 (Clontech) were provided by David Cortez. pLEGFP-C1 S123A, S129A, and S173A SMARCAL1 phosphomutants were generated using the QuikChange II XL site-directed mutagenesis kit (Agilent) and validated by Sanger sequencing. .. Using wt Ad5 E1B-55K and Ad12 E1B-55K cDNA templates, both Ad5 and Ad12 E1B-55K were amplified by PCR, digested with BamHI and XhoI, and subcloned into the pcDNA5/FRT/TO plasmid for the generation of tetracycline-inducible cell lines.

Isolation:

Article Title: Circ-ASH2L promotes tumor progression by sponging miR-34a to regulate Notch1 in pancreatic ductal adenocarcinoma
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Construct:

Article Title: The Human Cytomegalovirus Major Immediate-Early Distal Enhancer Region Is Required for Efficient Viral Replication and Immediate-Early Gene Expression
Article Snippet: .. The Hin dIII- Bam HI segment in each of the pΔ-300/-1108 SVgpt and pΔM SVgpt plasmids that contains the gpt ORF and SV40 intron and poly(A) site was replaced with the Hin dIII- Bam HI segment of phGFP-S65T (Clontech, Palo Alto, Calif.) that contains the green fluorescent protein ( gfp ) ORF and SV40 intron and poly(A) site, to produce pΔ-300/-1108 SVgfp and pΔ-640/-1108 SVgfp , respectively. pΔ-300/-640 SVgpt was constructed by replacing the corresponding Bam HI (blunted with T4 polymerase)- Sal I segment of pΔ-300/-1108 SVgfp with a corresponding Bsr GI (blunted with T4 polymerase)- Sal I fragment of pIE1EM. .. Plasmids pΔ-300/-1108 Egfp and pΔ-640/-1108 Egfp were derived from pΔ-300/-1108 SVgfp and pΔ-640/-1108 SVgfp , respectively, by replacing the entire basal early SV40 promoter with an adenovirus E1b TATA box.

Article Title: Adenovirus E1B 55-Kilodalton Protein Targets SMARCAL1 for Degradation during Infection and Modulates Cellular DNA Replication
Article Snippet: .. wt SMARCAL1 and ΔN-SMARCAL1 (lacking the N-terminal RPA interaction domain; ΔRPA) constructs cloned into the retroviral vector pLEGFP-C1 (Clontech) were provided by David Cortez. pLEGFP-C1 S123A, S129A, and S173A SMARCAL1 phosphomutants were generated using the QuikChange II XL site-directed mutagenesis kit (Agilent) and validated by Sanger sequencing. .. Using wt Ad5 E1B-55K and Ad12 E1B-55K cDNA templates, both Ad5 and Ad12 E1B-55K were amplified by PCR, digested with BamHI and XhoI, and subcloned into the pcDNA5/FRT/TO plasmid for the generation of tetracycline-inducible cell lines.

SYBR Green Assay:

Article Title: Serum regulates adipogenesis of mesenchymal stem cells via MEK/ERK-dependent PPARγ expression and phosphorylation
Article Snippet: .. Expression of PPARγ in MSCs was then quantified by real-time PCR by using the SYBR Green I PCR master mix (Takara, Dalian, China). .. Reactions were carried out on an ABI 7300 (Applied Biosystems, Shanghai, China) under the following conditions: cDNA was denatured for 15 min. at 94°C, followed by 40 cycles, consisting 30 sec. at 94°C, 30 sec. 58°C and 1 min. at 72°C.

Sequencing:

Article Title: Adenovirus E1B 55-Kilodalton Protein Targets SMARCAL1 for Degradation during Infection and Modulates Cellular DNA Replication
Article Snippet: .. wt SMARCAL1 and ΔN-SMARCAL1 (lacking the N-terminal RPA interaction domain; ΔRPA) constructs cloned into the retroviral vector pLEGFP-C1 (Clontech) were provided by David Cortez. pLEGFP-C1 S123A, S129A, and S173A SMARCAL1 phosphomutants were generated using the QuikChange II XL site-directed mutagenesis kit (Agilent) and validated by Sanger sequencing. .. Using wt Ad5 E1B-55K and Ad12 E1B-55K cDNA templates, both Ad5 and Ad12 E1B-55K were amplified by PCR, digested with BamHI and XhoI, and subcloned into the pcDNA5/FRT/TO plasmid for the generation of tetracycline-inducible cell lines.

Generated:

Article Title: Multiple Sequence Elements Facilitate Chp Rho GTPase Subcellular Location, Membrane Association, and Transforming Activity
Article Snippet: .. Mammalian Chp expression vectors were generated by fusing cDNA sequences encoding Chp, Chp[G40V], and ΔN-Chp[G40V] in frame with amino-terminal sequences encoding a hemagglutinin (HA) epitope tag in pBabe-puro-HAII retrovirus expression vector (a generous gift from Teresa Grana, University of North Carolina at Chapel Hill, Chapel Hill, NC) or in frame with amino-terminal sequences encoding green fluorescent protein (GFP) in the pEGFP-c3 expression vector (Clontech, Mountain View, CA). .. Further missense mutations were created by engineering the appropriate mutation during PCR-mediated DNA amplification to encode the 226Q, 228Q, 229Y, 230Q, and/or 231Q missense mutations in wild-type, activated (G40V), or activated and amino-terminally truncated [ΔN-Chp(40V]).

Article Title: Adenovirus E1B 55-Kilodalton Protein Targets SMARCAL1 for Degradation during Infection and Modulates Cellular DNA Replication
Article Snippet: .. wt SMARCAL1 and ΔN-SMARCAL1 (lacking the N-terminal RPA interaction domain; ΔRPA) constructs cloned into the retroviral vector pLEGFP-C1 (Clontech) were provided by David Cortez. pLEGFP-C1 S123A, S129A, and S173A SMARCAL1 phosphomutants were generated using the QuikChange II XL site-directed mutagenesis kit (Agilent) and validated by Sanger sequencing. .. Using wt Ad5 E1B-55K and Ad12 E1B-55K cDNA templates, both Ad5 and Ad12 E1B-55K were amplified by PCR, digested with BamHI and XhoI, and subcloned into the pcDNA5/FRT/TO plasmid for the generation of tetracycline-inducible cell lines.

Quantitative RT-PCR:

Article Title: Phosphoribosyl Pyrophosphate Amidotransferase Promotes the Progression of Thyroid Cancer via Regulating Pyruvate Kinase M2
Article Snippet: .. RNA Extraction and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) According to the manufacturer’s instructions, total RNA was extracted from TC tissues and cells using TRIzol (Invitrogen, Shanghai, China), and cDNA was prepared using the FastQuan RT kit (Tiangen Biotech., Beijing, China). qRT-PCR analysis was performed using SYBR Premix Ex Taq ™ Kit (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer’s instructions. .. Quantitative data were normalized to β-actin and the relative expression was calculated using the 2−ΔΔCt method.

Article Title: Circ-ASH2L promotes tumor progression by sponging miR-34a to regulate Notch1 in pancreatic ductal adenocarcinoma
Article Snippet: .. RNA isolation and qRT-PCR analysis Additionally, the miRNA mRQ 3′ primer was provided in the Mir-X miRNA qRT-PCR SYBR Kit (Clontech, Japan). .. Flow cytometric analysis The procedures of cell cycle analysis were referred to the manufacturer’s instructions of Cell Cycle Detection Kit (KGA512, KeyGEN BioTECH, China).

Article Title: Involvement of peroxisome proliferator-activated receptors in the estradiol production of ovine Sertoli cells
Article Snippet: .. The levels of PPARα, PPARβ, PPARγ, aromatase and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts were determined by qRT-PCR using SYBR® Premix Ex Taq™II (Takara Bio Inc., Otsu, Japan). ..

Article Title: Genome-wide identification and expression analysis of the NAC transcription factor family in tomato (Solanum lycopersicum) during aluminum stress
Article Snippet: .. For qRT-PCR analysis, one microgram of DNA-free RNA was transcribed into first strand cDNA by PrimeScriptTM RT Master Mix (TaKaRa). .. The qRT-PCR was carried out with the Roche LightCyler 480 instrument using SYBR Green chemistry (Toyobo).

Expressing:

Article Title: Multiple Sequence Elements Facilitate Chp Rho GTPase Subcellular Location, Membrane Association, and Transforming Activity
Article Snippet: .. Mammalian Chp expression vectors were generated by fusing cDNA sequences encoding Chp, Chp[G40V], and ΔN-Chp[G40V] in frame with amino-terminal sequences encoding a hemagglutinin (HA) epitope tag in pBabe-puro-HAII retrovirus expression vector (a generous gift from Teresa Grana, University of North Carolina at Chapel Hill, Chapel Hill, NC) or in frame with amino-terminal sequences encoding green fluorescent protein (GFP) in the pEGFP-c3 expression vector (Clontech, Mountain View, CA). .. Further missense mutations were created by engineering the appropriate mutation during PCR-mediated DNA amplification to encode the 226Q, 228Q, 229Y, 230Q, and/or 231Q missense mutations in wild-type, activated (G40V), or activated and amino-terminally truncated [ΔN-Chp(40V]).

Article Title: Serum regulates adipogenesis of mesenchymal stem cells via MEK/ERK-dependent PPARγ expression and phosphorylation
Article Snippet: .. Expression of PPARγ in MSCs was then quantified by real-time PCR by using the SYBR Green I PCR master mix (Takara, Dalian, China). .. Reactions were carried out on an ABI 7300 (Applied Biosystems, Shanghai, China) under the following conditions: cDNA was denatured for 15 min. at 94°C, followed by 40 cycles, consisting 30 sec. at 94°C, 30 sec. 58°C and 1 min. at 72°C.

Polymerase Chain Reaction:

Article Title: Serum regulates adipogenesis of mesenchymal stem cells via MEK/ERK-dependent PPARγ expression and phosphorylation
Article Snippet: .. Expression of PPARγ in MSCs was then quantified by real-time PCR by using the SYBR Green I PCR master mix (Takara, Dalian, China). .. Reactions were carried out on an ABI 7300 (Applied Biosystems, Shanghai, China) under the following conditions: cDNA was denatured for 15 min. at 94°C, followed by 40 cycles, consisting 30 sec. at 94°C, 30 sec. 58°C and 1 min. at 72°C.

Real-time Polymerase Chain Reaction:

Article Title: Phosphoribosyl Pyrophosphate Amidotransferase Promotes the Progression of Thyroid Cancer via Regulating Pyruvate Kinase M2
Article Snippet: .. RNA Extraction and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) According to the manufacturer’s instructions, total RNA was extracted from TC tissues and cells using TRIzol (Invitrogen, Shanghai, China), and cDNA was prepared using the FastQuan RT kit (Tiangen Biotech., Beijing, China). qRT-PCR analysis was performed using SYBR Premix Ex Taq ™ Kit (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer’s instructions. .. Quantitative data were normalized to β-actin and the relative expression was calculated using the 2−ΔΔCt method.

Article Title: Serum regulates adipogenesis of mesenchymal stem cells via MEK/ERK-dependent PPARγ expression and phosphorylation
Article Snippet: .. Expression of PPARγ in MSCs was then quantified by real-time PCR by using the SYBR Green I PCR master mix (Takara, Dalian, China). .. Reactions were carried out on an ABI 7300 (Applied Biosystems, Shanghai, China) under the following conditions: cDNA was denatured for 15 min. at 94°C, followed by 40 cycles, consisting 30 sec. at 94°C, 30 sec. 58°C and 1 min. at 72°C.

Recombinase Polymerase Amplification:

Article Title: Adenovirus E1B 55-Kilodalton Protein Targets SMARCAL1 for Degradation during Infection and Modulates Cellular DNA Replication
Article Snippet: .. wt SMARCAL1 and ΔN-SMARCAL1 (lacking the N-terminal RPA interaction domain; ΔRPA) constructs cloned into the retroviral vector pLEGFP-C1 (Clontech) were provided by David Cortez. pLEGFP-C1 S123A, S129A, and S173A SMARCAL1 phosphomutants were generated using the QuikChange II XL site-directed mutagenesis kit (Agilent) and validated by Sanger sequencing. .. Using wt Ad5 E1B-55K and Ad12 E1B-55K cDNA templates, both Ad5 and Ad12 E1B-55K were amplified by PCR, digested with BamHI and XhoI, and subcloned into the pcDNA5/FRT/TO plasmid for the generation of tetracycline-inducible cell lines.

Plasmid Preparation:

Article Title: Multiple Sequence Elements Facilitate Chp Rho GTPase Subcellular Location, Membrane Association, and Transforming Activity
Article Snippet: .. Mammalian Chp expression vectors were generated by fusing cDNA sequences encoding Chp, Chp[G40V], and ΔN-Chp[G40V] in frame with amino-terminal sequences encoding a hemagglutinin (HA) epitope tag in pBabe-puro-HAII retrovirus expression vector (a generous gift from Teresa Grana, University of North Carolina at Chapel Hill, Chapel Hill, NC) or in frame with amino-terminal sequences encoding green fluorescent protein (GFP) in the pEGFP-c3 expression vector (Clontech, Mountain View, CA). .. Further missense mutations were created by engineering the appropriate mutation during PCR-mediated DNA amplification to encode the 226Q, 228Q, 229Y, 230Q, and/or 231Q missense mutations in wild-type, activated (G40V), or activated and amino-terminally truncated [ΔN-Chp(40V]).

Article Title: Adenovirus E1B 55-Kilodalton Protein Targets SMARCAL1 for Degradation during Infection and Modulates Cellular DNA Replication
Article Snippet: .. wt SMARCAL1 and ΔN-SMARCAL1 (lacking the N-terminal RPA interaction domain; ΔRPA) constructs cloned into the retroviral vector pLEGFP-C1 (Clontech) were provided by David Cortez. pLEGFP-C1 S123A, S129A, and S173A SMARCAL1 phosphomutants were generated using the QuikChange II XL site-directed mutagenesis kit (Agilent) and validated by Sanger sequencing. .. Using wt Ad5 E1B-55K and Ad12 E1B-55K cDNA templates, both Ad5 and Ad12 E1B-55K were amplified by PCR, digested with BamHI and XhoI, and subcloned into the pcDNA5/FRT/TO plasmid for the generation of tetracycline-inducible cell lines.

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    TaKaRa adenovirus e1b promoter
    PAX4 repressor domains. (A) Comparison of PAX4 and PAX6 structure. PD, paired domain; HD, homeodomain. (B) Transient transfections of the indicated cell lines were performed with the chimeric PAX4-GAL4 DBD fusion protein constructs shown and a reporter construct consisting of the GAL4 binding site linked to HSV-TK driving CAT expression (GAL4UAS-TK-CAT). (C) Transient transfections of βTC3 and NIH3T3 cells were performed with a reporter construct consisting of five copies of the GAL4 GAL4 UAS. (D) Transient transfections of αTC1.6 cells were performed with a reporter construct consisting of HSV-TK driving CAT expression without a GAL4 binding site (TK-CAT). (E) Transient transfections of βTC3 cells were performed with a reporter consisting of five GAL4 binding sites linked to the minimal adenovirus <t>E1b</t> promoter driving CAT expression (5XGAL4UAS-E1b-CAT). Note that the lowermost expression construct consists of the PAX6 C-terminal domain fused to the GAL4 DBD. Transfections were performed in duplicate on at least three separate occasions. Relative CAT activity of the GAL4-DBD expression construct alone is set arbitrarily at +1. Graphs show mean ± standard error of the mean.
    Adenovirus E1b Promoter, supplied by TaKaRa, used in various techniques. Bioz Stars score: 80/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    TaKaRa ad e1b 55k expression
    SMARCAL1 is targeted for degradation during Ad infection. A549 cells were either mock infected or infected with 10 PFU/cell of wt Ad5 or wt Ad12 and harvested at the appropriate times postinfection. (A) Ad5 cell lysates were then subjected to WB for SMARCAL1, p53, <t>E1B-55K,</t> E4orf6, and β-actin. (B) Ad12 cell lysates were subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of more than three independent experiments.
    Ad E1b 55k Expression, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    TaKaRa adenovirus e1b promoter tataa box
    Diagram of the GFP-reporter mammalian two-hybrid system with extrachromosomal maintenance of prey expression plasmid. GB133 cells, which express the EBNA-1 protein, harbor a GAL4-dependent GFP reporter plasmid in their chromosomes. For interaction screening, GB133 cells can be stably transfected with an expression plasmid for a GAL4 DNA-binding domain (GAL4DB) fusion protein of bait. Cells can then be transiently transfected with a cDNA library expressing fusion proteins of a transcriptional activating domain (AD) and preys. The two-hybrid interaction will induce transcription of the GFP gene, which is readily detectable by fluorescent microscopy of living cells. Once introduced into GB133 cells by transient transfection protocols, the prey-expressing plasmids harboring the OriP replication origin sequence will be maintained stably because of the presence of the EBNA-1 protein, thus keeping the positive prey-expressing cells permanently green fluorescent. GAL4BE, GAL4 binding element; TATA , <t>TATAA</t> box from adenovirus <t>E1b</t> gene promoter.
    Adenovirus E1b Promoter Tataa Box, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PAX4 repressor domains. (A) Comparison of PAX4 and PAX6 structure. PD, paired domain; HD, homeodomain. (B) Transient transfections of the indicated cell lines were performed with the chimeric PAX4-GAL4 DBD fusion protein constructs shown and a reporter construct consisting of the GAL4 binding site linked to HSV-TK driving CAT expression (GAL4UAS-TK-CAT). (C) Transient transfections of βTC3 and NIH3T3 cells were performed with a reporter construct consisting of five copies of the GAL4 GAL4 UAS. (D) Transient transfections of αTC1.6 cells were performed with a reporter construct consisting of HSV-TK driving CAT expression without a GAL4 binding site (TK-CAT). (E) Transient transfections of βTC3 cells were performed with a reporter consisting of five GAL4 binding sites linked to the minimal adenovirus E1b promoter driving CAT expression (5XGAL4UAS-E1b-CAT). Note that the lowermost expression construct consists of the PAX6 C-terminal domain fused to the GAL4 DBD. Transfections were performed in duplicate on at least three separate occasions. Relative CAT activity of the GAL4-DBD expression construct alone is set arbitrarily at +1. Graphs show mean ± standard error of the mean.

    Journal: Molecular and Cellular Biology

    Article Title: Paired-Homeodomain Transcription Factor PAX4 Acts as a Transcriptional Repressor in Early Pancreatic Development

    doi:

    Figure Lengend Snippet: PAX4 repressor domains. (A) Comparison of PAX4 and PAX6 structure. PD, paired domain; HD, homeodomain. (B) Transient transfections of the indicated cell lines were performed with the chimeric PAX4-GAL4 DBD fusion protein constructs shown and a reporter construct consisting of the GAL4 binding site linked to HSV-TK driving CAT expression (GAL4UAS-TK-CAT). (C) Transient transfections of βTC3 and NIH3T3 cells were performed with a reporter construct consisting of five copies of the GAL4 GAL4 UAS. (D) Transient transfections of αTC1.6 cells were performed with a reporter construct consisting of HSV-TK driving CAT expression without a GAL4 binding site (TK-CAT). (E) Transient transfections of βTC3 cells were performed with a reporter consisting of five GAL4 binding sites linked to the minimal adenovirus E1b promoter driving CAT expression (5XGAL4UAS-E1b-CAT). Note that the lowermost expression construct consists of the PAX6 C-terminal domain fused to the GAL4 DBD. Transfections were performed in duplicate on at least three separate occasions. Relative CAT activity of the GAL4-DBD expression construct alone is set arbitrarily at +1. Graphs show mean ± standard error of the mean.

    Article Snippet: To ensure that the transcriptional activation potential of PAX4 was not hidden by the high basal activity of the HSV-TK promoter, we also tested the same fusion constructs with a construct containing five GAL4 UASs linked to the adenovirus E1b promoter driving CAT expression (pG5CAT; Clontech).

    Techniques: Transfection, Construct, Binding Assay, Expressing, Activity Assay

    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.

    Journal: Molecular Biology of the Cell

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

    doi:

    Figure Lengend 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.

    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).

    Techniques: 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.

    Journal: Molecular Biology of the Cell

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

    doi:

    Figure Lengend 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.

    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).

    Techniques: Expressing, Plasmid Preparation, Transfection

    SMARCAL1 is targeted for degradation during Ad infection. A549 cells were either mock infected or infected with 10 PFU/cell of wt Ad5 or wt Ad12 and harvested at the appropriate times postinfection. (A) Ad5 cell lysates were then subjected to WB for SMARCAL1, p53, E1B-55K, E4orf6, and β-actin. (B) Ad12 cell lysates were subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of more than three independent experiments.

    Journal: Journal of Virology

    Article Title: Adenovirus E1B 55-Kilodalton Protein Targets SMARCAL1 for Degradation during Infection and Modulates Cellular DNA Replication

    doi: 10.1128/JVI.00402-19

    Figure Lengend Snippet: SMARCAL1 is targeted for degradation during Ad infection. A549 cells were either mock infected or infected with 10 PFU/cell of wt Ad5 or wt Ad12 and harvested at the appropriate times postinfection. (A) Ad5 cell lysates were then subjected to WB for SMARCAL1, p53, E1B-55K, E4orf6, and β-actin. (B) Ad12 cell lysates were subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of more than three independent experiments.

    Article Snippet: Cells were then fed every 3 days; individual colonies were ultimately selected, expanded, and assessed for Ad E1B-55K expression following incubation with 0.1 μg/ml doxycycline for 24 h. To generate GFP-SMARCAL1 cell lines, pLEGFP-C1 SMARCAL1 constructs were transfected in a 1:1 ratio with the pVSV envelope plasmid in the GP2-293 retrovirus packaging cell line (Clontech) using Lipofectamine 2000.

    Techniques: Infection, Western Blot

    SMARCAL1 is degraded during Ad infection in an E1B-55K/E4orf6- and CRL-dependent manner. (A) A549 cells were either mock infected, infected with wt Ad5, or infected with E1B-55K ( dl 1520), E4orf3 (H5 pm 4150), or E4orf6 (H5 pm 4154) deletion virus. At 24 h and 48 h postinfection, cells were harvested and subjected to WB for SMARCAL1, p53, E1B-55K, E4orf3, E4orf6, and β-actin. (B) A549 cells were either mock infected, infected with wt Ad12, or infected with the E1B-55K ( dl 620) deletion virus. At 24 h and 48 h postinfection, cells were harvested and Western blotted for SMARCAL1, p53, E1B-55K, and β-actin. (C and D) A549 cells were either mock infected or infected with wt Ad5 or wt Ad12 in the absence or presence of 100 nM or 500 nM MLN4924. At 24 h and 48 h postinfection, cells were harvested and subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of three independent experiments.

    Journal: Journal of Virology

    Article Title: Adenovirus E1B 55-Kilodalton Protein Targets SMARCAL1 for Degradation during Infection and Modulates Cellular DNA Replication

    doi: 10.1128/JVI.00402-19

    Figure Lengend Snippet: SMARCAL1 is degraded during Ad infection in an E1B-55K/E4orf6- and CRL-dependent manner. (A) A549 cells were either mock infected, infected with wt Ad5, or infected with E1B-55K ( dl 1520), E4orf3 (H5 pm 4150), or E4orf6 (H5 pm 4154) deletion virus. At 24 h and 48 h postinfection, cells were harvested and subjected to WB for SMARCAL1, p53, E1B-55K, E4orf3, E4orf6, and β-actin. (B) A549 cells were either mock infected, infected with wt Ad12, or infected with the E1B-55K ( dl 620) deletion virus. At 24 h and 48 h postinfection, cells were harvested and Western blotted for SMARCAL1, p53, E1B-55K, and β-actin. (C and D) A549 cells were either mock infected or infected with wt Ad5 or wt Ad12 in the absence or presence of 100 nM or 500 nM MLN4924. At 24 h and 48 h postinfection, cells were harvested and subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of three independent experiments.

    Article Snippet: Cells were then fed every 3 days; individual colonies were ultimately selected, expanded, and assessed for Ad E1B-55K expression following incubation with 0.1 μg/ml doxycycline for 24 h. To generate GFP-SMARCAL1 cell lines, pLEGFP-C1 SMARCAL1 constructs were transfected in a 1:1 ratio with the pVSV envelope plasmid in the GP2-293 retrovirus packaging cell line (Clontech) using Lipofectamine 2000.

    Techniques: Infection, Western Blot

    Generation and characterization of tetracycline-inducible Ad5 and Ad12 E1B-55K FlpIn U2OS cells. FlpIn U2OS cells were transfected with Ad5 E1B-55K and Ad12 E1B-55K pcDNA5/FRT/TO plasmids and the recombination plasmid pOG44. Cells were incubated in selection medium containing hygromycin (200 μg/ml). Individual colonies were isolated, expanded, and treated with 0.1 μg/ml doxycycline. Twenty-four h postinduction, cell lysates were harvested, separated by SDS-PAGE, and subjected to WB analysis for Ad5 and Ad12 E1B-55K. WB analyses were also performed to gauge the levels of SMARCAL1, p53, MRE11, and β-actin for Ad5 E1B-55K and Ad12 E1B-55K FlpIn U2OS cells. Data are representative of more than three independent experiments.

    Journal: Journal of Virology

    Article Title: Adenovirus E1B 55-Kilodalton Protein Targets SMARCAL1 for Degradation during Infection and Modulates Cellular DNA Replication

    doi: 10.1128/JVI.00402-19

    Figure Lengend Snippet: Generation and characterization of tetracycline-inducible Ad5 and Ad12 E1B-55K FlpIn U2OS cells. FlpIn U2OS cells were transfected with Ad5 E1B-55K and Ad12 E1B-55K pcDNA5/FRT/TO plasmids and the recombination plasmid pOG44. Cells were incubated in selection medium containing hygromycin (200 μg/ml). Individual colonies were isolated, expanded, and treated with 0.1 μg/ml doxycycline. Twenty-four h postinduction, cell lysates were harvested, separated by SDS-PAGE, and subjected to WB analysis for Ad5 and Ad12 E1B-55K. WB analyses were also performed to gauge the levels of SMARCAL1, p53, MRE11, and β-actin for Ad5 E1B-55K and Ad12 E1B-55K FlpIn U2OS cells. Data are representative of more than three independent experiments.

    Article Snippet: Cells were then fed every 3 days; individual colonies were ultimately selected, expanded, and assessed for Ad E1B-55K expression following incubation with 0.1 μg/ml doxycycline for 24 h. To generate GFP-SMARCAL1 cell lines, pLEGFP-C1 SMARCAL1 constructs were transfected in a 1:1 ratio with the pVSV envelope plasmid in the GP2-293 retrovirus packaging cell line (Clontech) using Lipofectamine 2000.

    Techniques: Transfection, Plasmid Preparation, Incubation, Selection, Isolation, SDS Page, Western Blot

    Ad E1B-55K associates with SMARCAL1 in Ad-transformed cells. (A) Ad E1B-55K and SMARCAL1 were immunoprecipitated from Ad5 HEK 293 cells (A) and Ad12 HER2 cells (B) and subjected to WB for E1B-55K and SMARCAL1. IgG, immunoglobulin control IP.

    Journal: Journal of Virology

    Article Title: Adenovirus E1B 55-Kilodalton Protein Targets SMARCAL1 for Degradation during Infection and Modulates Cellular DNA Replication

    doi: 10.1128/JVI.00402-19

    Figure Lengend Snippet: Ad E1B-55K associates with SMARCAL1 in Ad-transformed cells. (A) Ad E1B-55K and SMARCAL1 were immunoprecipitated from Ad5 HEK 293 cells (A) and Ad12 HER2 cells (B) and subjected to WB for E1B-55K and SMARCAL1. IgG, immunoglobulin control IP.

    Article Snippet: Cells were then fed every 3 days; individual colonies were ultimately selected, expanded, and assessed for Ad E1B-55K expression following incubation with 0.1 μg/ml doxycycline for 24 h. To generate GFP-SMARCAL1 cell lines, pLEGFP-C1 SMARCAL1 constructs were transfected in a 1:1 ratio with the pVSV envelope plasmid in the GP2-293 retrovirus packaging cell line (Clontech) using Lipofectamine 2000.

    Techniques: Transformation Assay, Immunoprecipitation, Western Blot

    ATR kinase and CDKs promote SMARCAL1 degradation following Ad5 and Ad12 infection. A549 cells were either mock infected or infected with 10 PFU/cell of wt Ad5 (A and C) or wt Ad12 (B and D). Cells were then incubated in the absence or presence of ATR inhibitor (AZD6738 [ATRi], 1 μM; A and B) or ATR and CDK inhibitors (AZD6738, 1 μM and RO-3306 [CDKi], 9 μM; C and D) and harvested at the appropriate times postinfection. Cell lysates were then separated by SDS-PAGE and subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of three independent experiments.

    Journal: Journal of Virology

    Article Title: Adenovirus E1B 55-Kilodalton Protein Targets SMARCAL1 for Degradation during Infection and Modulates Cellular DNA Replication

    doi: 10.1128/JVI.00402-19

    Figure Lengend Snippet: ATR kinase and CDKs promote SMARCAL1 degradation following Ad5 and Ad12 infection. A549 cells were either mock infected or infected with 10 PFU/cell of wt Ad5 (A and C) or wt Ad12 (B and D). Cells were then incubated in the absence or presence of ATR inhibitor (AZD6738 [ATRi], 1 μM; A and B) or ATR and CDK inhibitors (AZD6738, 1 μM and RO-3306 [CDKi], 9 μM; C and D) and harvested at the appropriate times postinfection. Cell lysates were then separated by SDS-PAGE and subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of three independent experiments.

    Article Snippet: Cells were then fed every 3 days; individual colonies were ultimately selected, expanded, and assessed for Ad E1B-55K expression following incubation with 0.1 μg/ml doxycycline for 24 h. To generate GFP-SMARCAL1 cell lines, pLEGFP-C1 SMARCAL1 constructs were transfected in a 1:1 ratio with the pVSV envelope plasmid in the GP2-293 retrovirus packaging cell line (Clontech) using Lipofectamine 2000.

    Techniques: Infection, Incubation, SDS Page, Western Blot

    Ad5 and Ad12 E1B-55K modulate cellular DNA replication rates and promote replication fork stalling. Uninduced and doxycycline-induced Ad5 and Ad12 E1B-55K FlpIn U2OS cells were labeled with 25 μM CldU and 250 μM IdU for 20 min each. DNA fiber spreads were then prepared and denatured with 2.5 M HCl. DNA fibers were labeled with the appropriate primary and secondary antibodies and visualized using a Nikon E600 microscope. (A and B) Representative DNA spreads (with or without Ad5 or Ad12 E1B-55K) are shown indicating the mean fork speeds; CldU and IdU fork lengths were quantified and presented as dot plots (± standard deviations [SD]), with the mean fork speed shown as a red bar. n = 3 (total fibers analyzed: Ad5 mock infected, 347; Ad5 E1B-55K, 368; Ad12 mock infected, 370; Ad12 E1B-55K, 364). (C) Percent stalled forks (CldU-only labeled forks) were quantified and presented as a bar chart (±SD). In all instances data presented were subjected to analysis of variance with two-tailed t test: Ad5 E1B-55K CldU tract length relative to the mock CldU tract length, P = 4.8E−20 (***); Ad5 E1B-55K CldU/IdU ratio relative to the mock CldU tract length, P = 9.44E−45 (****); Ad12 E1B-55K CldU tract length relative to the mock CldU tract length, P = 1.29E−32 (****); Ad12 E1B-55K CldU/IdU ratio relative to the mock CldU tract length, P = 6.32E−61 (****); ns, not significant. For significance for stalled forks, Ad5 E1B-55K relative to mock infection, P = 0.009 (**); Ad12 E1B-55K relative mock infection, P = 0.002 (**).

    Journal: Journal of Virology

    Article Title: Adenovirus E1B 55-Kilodalton Protein Targets SMARCAL1 for Degradation during Infection and Modulates Cellular DNA Replication

    doi: 10.1128/JVI.00402-19

    Figure Lengend Snippet: Ad5 and Ad12 E1B-55K modulate cellular DNA replication rates and promote replication fork stalling. Uninduced and doxycycline-induced Ad5 and Ad12 E1B-55K FlpIn U2OS cells were labeled with 25 μM CldU and 250 μM IdU for 20 min each. DNA fiber spreads were then prepared and denatured with 2.5 M HCl. DNA fibers were labeled with the appropriate primary and secondary antibodies and visualized using a Nikon E600 microscope. (A and B) Representative DNA spreads (with or without Ad5 or Ad12 E1B-55K) are shown indicating the mean fork speeds; CldU and IdU fork lengths were quantified and presented as dot plots (± standard deviations [SD]), with the mean fork speed shown as a red bar. n = 3 (total fibers analyzed: Ad5 mock infected, 347; Ad5 E1B-55K, 368; Ad12 mock infected, 370; Ad12 E1B-55K, 364). (C) Percent stalled forks (CldU-only labeled forks) were quantified and presented as a bar chart (±SD). In all instances data presented were subjected to analysis of variance with two-tailed t test: Ad5 E1B-55K CldU tract length relative to the mock CldU tract length, P = 4.8E−20 (***); Ad5 E1B-55K CldU/IdU ratio relative to the mock CldU tract length, P = 9.44E−45 (****); Ad12 E1B-55K CldU tract length relative to the mock CldU tract length, P = 1.29E−32 (****); Ad12 E1B-55K CldU/IdU ratio relative to the mock CldU tract length, P = 6.32E−61 (****); ns, not significant. For significance for stalled forks, Ad5 E1B-55K relative to mock infection, P = 0.009 (**); Ad12 E1B-55K relative mock infection, P = 0.002 (**).

    Article Snippet: Cells were then fed every 3 days; individual colonies were ultimately selected, expanded, and assessed for Ad E1B-55K expression following incubation with 0.1 μg/ml doxycycline for 24 h. To generate GFP-SMARCAL1 cell lines, pLEGFP-C1 SMARCAL1 constructs were transfected in a 1:1 ratio with the pVSV envelope plasmid in the GP2-293 retrovirus packaging cell line (Clontech) using Lipofectamine 2000.

    Techniques: Labeling, Microscopy, Infection, Two Tailed Test

    Diagram of the GFP-reporter mammalian two-hybrid system with extrachromosomal maintenance of prey expression plasmid. GB133 cells, which express the EBNA-1 protein, harbor a GAL4-dependent GFP reporter plasmid in their chromosomes. For interaction screening, GB133 cells can be stably transfected with an expression plasmid for a GAL4 DNA-binding domain (GAL4DB) fusion protein of bait. Cells can then be transiently transfected with a cDNA library expressing fusion proteins of a transcriptional activating domain (AD) and preys. The two-hybrid interaction will induce transcription of the GFP gene, which is readily detectable by fluorescent microscopy of living cells. Once introduced into GB133 cells by transient transfection protocols, the prey-expressing plasmids harboring the OriP replication origin sequence will be maintained stably because of the presence of the EBNA-1 protein, thus keeping the positive prey-expressing cells permanently green fluorescent. GAL4BE, GAL4 binding element; TATA , TATAA box from adenovirus E1b gene promoter.

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

    Article Title: A green fluorescent protein-reporter mammalian two-hybrid system with extrachromosomal maintenance of a prey expression plasmid: Application to interaction screening

    doi:

    Figure Lengend Snippet: Diagram of the GFP-reporter mammalian two-hybrid system with extrachromosomal maintenance of prey expression plasmid. GB133 cells, which express the EBNA-1 protein, harbor a GAL4-dependent GFP reporter plasmid in their chromosomes. For interaction screening, GB133 cells can be stably transfected with an expression plasmid for a GAL4 DNA-binding domain (GAL4DB) fusion protein of bait. Cells can then be transiently transfected with a cDNA library expressing fusion proteins of a transcriptional activating domain (AD) and preys. The two-hybrid interaction will induce transcription of the GFP gene, which is readily detectable by fluorescent microscopy of living cells. Once introduced into GB133 cells by transient transfection protocols, the prey-expressing plasmids harboring the OriP replication origin sequence will be maintained stably because of the presence of the EBNA-1 protein, thus keeping the positive prey-expressing cells permanently green fluorescent. GAL4BE, GAL4 binding element; TATA , TATAA box from adenovirus E1b gene promoter.

    Article Snippet: An expression plasmid for MSG1 harboring the OriP sequence was constructed by inserting hemagglutinin-tagged human MSG1 cDNA ( ) into the multiple cloning site of pCMV.OriP. pG5GFP, a GAL4-dependent reporter plasmid for expression of the green fluorescent protein (GFP), contained five repeats of GAL4 binding elements followed by the adenovirus E1b promoter/TATAA box and GFP cDNA; it was constructed by replacing the chloramphenicol acetyltransferase cDNA cassette of pG5CAT (CLONTECH) with a humanized GFP cDNA excised from pEGFP (CLONTECH).

    Techniques: Expressing, Plasmid Preparation, Stable Transfection, Transfection, Binding Assay, cDNA Library Assay, Microscopy, Sequencing