Notes: To study PHB-mediated gene repression and cell cycle arrest we generated promoter-reporter fusions using the pGL4 Luciferase Vectors. The PHB-E2F1 interaction was investigated using the CheckMate™ Mammalian two-hybrid system. The NanoBiT™ PPI interaction system was used for additional analysis of the PHB-E2F1 interaction. cDNA was synthesized using the Reverse Transcription System. (4900)

Notes: This paper examined the role of the Cdk3/c-Jun signaling in EGF-stimulated cell proliferation and cell transformation. An AP-1 luciferase reporter plasmid construct was contransfected with various expression vector combinations of Cdk3, Cdk2, c-Jun, c-Jun M63/73, Cdk3-DN and cyclin C and the phRL-SV40 Vector as a normalization control. Cells were lysed at room temperature for 30 minutes with gentle shaking and luciferase activity measured using the Dual-Luciferase^® Reporter Assay System. SaoS-2 cells transfected with a mock and Cdk3 RNAi vector were seeded at a density of 4 x 10³ in 96-well plates with 20µl of medium. After 24 hours, 20µl of CellTiter^® 96 AQ_ueous One Solution was dispensed to each well, and the plate incubated for 1 hour at 37°C. The reaction was stopped using 25µl of 10% SDS and absorbance was measured at 492 and 690nm. For the CheckMate™ Mammalian Two-Hybrid System, the plasmid constructs, pACT-c-Jun, pBIND-Cdk3 and pG5luc, were cotransfected into HEK293 cells at no more than 100ng/well in a 48-well plate. After incubation and lysis, 20µl of lysate was dispensed into each well of a 96-well luminescence plate. Firefly and Renilla luciferase activity was detected. (4028)

Notes: The authors were interested in examined the relationship between pregnane X receptor (PXR) and protein arginine methyltransferase 1 (PRMT1). Cells were transiently transfected for 60 hours including chemical treatment, and the Luciferase Assay System was used to analyze reporter activity. For the Checkmate™ Mammalian Two-Hybrid Assay, CV-1 cells were transfected with the pBIND Vector containing PXR, pACT Vector containing PRMT1 and pG5luc Vector. After 12 hours, the cells were treated with rifampicin and 48 hours later, luciferase activity was measured. Full-length PRMT1 protein was synthesized using the TNT^® SP6 Coupled Reticulocyte Lysate System and used in a GST pulldown assay. (4027)

Notes: The CheckMate™ Mammalian Two-Hybrid System was used to explore the dimerization of epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases without ligand. The intracellular domains of EGFR, ErbB2, ErbB3 and ErbB4 were amplified and cloned into both the pACT and pBIND Vectors. Transfection into NIH3T3 cells in 12-well plates occurred with 0.3 μg of pG5luc Vector (the reporter vector), 0.2 μg of pACT Vector or an equimolar amount of pACT construct, and 0.1 μg of pBIND Vector or an equimolar amount of pBIND construct. After 24 hours, the cells were lysed and luciferase activity assessed using the Dual-Luciferase® Reporter Assay System. (3993)

Notes: To examine the role of cyclin-dependent kinase (cdk)-3 expression in cancer cell lines, potential targets of cdk3 phosphorylation were examined. The CheckMate™ Mammalian Two-Hybrid System was used to test for transcription factor binding partners of cdk in HEK293 cells. Cell proliferation of T96G cells stably transfected with either cdk3 or vector was assessed using the CellTiter 96® AQueous One Solution Cell Proliferation Assay. (3983)

Notes: To learn about the ideal target binding sequence for SATB1, the T-lineage-enriched chromatin organizer and transcription factor, random oligonucleotides underwent SELEX and five rounds of selection by EMSA. The enriched library of oligos was cloned into the pGEM®-T Easy Vector, transformed and sequenced. Several variants of SATB1-binding consensus sequences were annealed, ligated into the pGL3-Promoter Vector and cotransfected into HEK 293 cells with a plasmid that either contained SATB1 or was empty. After 48 hours, the cells were harvested and luciferase activity measured. The CheckMate™ Mammalian Two-Hybrid System was used to assess how the N-terminal PDZ domain of SATB1 interacted with the Cut and homeodomain in the C-terminus. (3982)

Notes: This article examines the interaction between CAR, a member of the nuclear steroid/thyroid hormone receptor family, which translocates from the cytoplasm to the nucleus when cells are exposed to phenobaritol, and the membrane-associated subunit of protein phosphatase 1 (PPP1R16A, or abbreviated as R16A), which is a novel CAR-binding protein. The R16A protein, it was expressed using the TnT^® Coupled Reticulocyte Lysate System and labeled with ³⁵S. This protein was incubated with GST-hCAR-fusion protein attached to a glutathione resin; the resin was washed, and the bound protein was separated by PAGE and detected by autoradiography. Affinity-tagged R16A was expressed in HepG2 cells, purified and separated by SDS-PAGE. The two major bands were excised, digested with Sequencing Grade Modified Trypsin, lyophilized, resuspended in acetonitrile and subjected to mass spectrometric analyses. The CheckMate™ Mammalian Two-Hybrid System was used to examine the interaction between wildtype and mutated R16A. Forty-eight hours posttransfection into HepG2 cells or 16 hours after DNA injection into mice and liver homogenization, the luciferase activities were measured using the Dual-Luciferase^® Reporter Assay System. (3752)

Notes: These authors used a proteomics-based approach to isolate and identify proteins that interact with the estrogen receptor ERα. The protein DBC-1 was identified as a ligand-independent binding partner of ERα. The amino terminus of DBC-1 bound directly to the ERα hormone binding domain in the absence of estradiol. Initial identification of DBC-1 was based on peptide sequence analysis of proteins that coimmunoprecipitated with unliganded ERα. The CheckMate™ Mammalian Two-Hybrid System was then used to confirm the interaction. DBC-1 was fused to the GAL4-DNA-binding domain in the pBIND vector, and ERα was fused to the VP16 activation domain in the pACT vector. These proteins were then expressed independently or together in HeLa cells and their ability to activate transcription of a luciferase reporter controlled by GAL-4-DNA binding sites (pG5luc) was evaluated in both the presence and absence of estradiol. Further analysis with truncated proteins was used to demonstrate that the interaction between DBC-1 and ERα was mediated by the amino terminal part of the DBC-1 protein. (3628)

Notes: Dimerization of HIV-1 protease subunuts is essential for proteolytic activity and viral replication. These authors used a FRET assay to identify potential small molecule inhibitors of HIV-1 protease dimerization. After identifying a number of inhibitors in the FRET assay, they used the CheckMate™ Mammalian Two-Hybrid System to independently confirm the disruption of interaction between the two protein subunits. (3711)

Notes: The authors wanted to screen inhibitory compounds for the HIV-1 accessory protein Nef using both computer modeling and experimental methods. Using a structure-based program for the SH3 binding surface of Nef, drug compounds were screened in silico and then further analyzed using a cell-based assay. The Nef gene and SH3 domain were cloned into the pACT and pBIND Vectors of the CheckMate™ Mammalian Two-Hybrid System, transfected into COS-7 cells, and 18 hours later, the cells exposed to potential inhibitors. After 24 hours, luciferase activity was assessed using either the Dual-Glo™ or the Steady-Glo^® Luciferase Assay Systems. (3751)

Notes: The CheckMate™ Mammalian Two-Hybrid System was used to screen for protein-binding partners for RSK2, a ribosomal S6 kinase involved in myoblast differentiation. The RSK2 cDNA was cloned into the pBIND Vector as bait while several transcription factors were amplified by PCR and cloned into the pACT Vector. The bait pBIND-RSK2 construct, the pACT-transcription factors and pG5-luc Vector were transfected into 293 cells at a 1:1:1 molar ratio. To assess the protein interaction, the cells were lysed, and the firefly luciferase activity was normalized to Renilla luciferase activity. The strongest interaction was with NFAT3. (3574)

Notes: To examine the putative binding of the poliovirus nonstructural proteins to each other, the wildtype and mutant 2B, 2C, 2BC, 3A, and 3AB regions were amplified and cloned into the CheckMate™ Mammalian Two-Hybrid System vectors, pACT and pBIND, after digestion with Sal I and Mlu I. COS cells grown in 12-well plates were transfected with a total of 1.6µg DNA. To reduce competition for transcription factors, the mix of pBIND:pACT:pG5luc plasmids was adjusted from 1:1:1 to 0.053µg pBIND, 0.053µg pACT with a higher concentration of pG5luc (1µg) and an additional 0.49µg of carrier pGEM^®-3. After 24 to 28 hours, the firefly luciferase activity was assessed using 10µl of cell lysate with 100µl Luciferase Assay Reagent and measuring the luminescence. (3492)

Notes: The CheckMate™ Mammalian Two-Hybrid System was used to study the association of Lyn, a tyrosine kinase, and flotillin-1, a novel constituent of lipid rafts. NIH/3T3 cells were seeded in 60mm dishes at a density of ~1 × 10⁵ cells. Each plate of cells was transfected with a total of 5.4µg of plasmid DNA (a 1:1:1 mixture of pBIND-flotillin-1 vector, pACT-Lyn vector, and pG5luc vector) by lipofection. Both mutant and wildtype proteins were used to assess the interaction. The cells were harvested, lyses and assessed for firefly and Renilla luciferase activities using the Dual-Luciferase^® Reporter Assay System. The results were expressed as a ratio of firefly to Renilla luciferase activity. (3491)

Notes: To test the ability of SorICD to stimulate transcription of a Gal4-dependent luciferase promoter, COS-7 cells were transfected with pG5luc, pBIND, or pBINDSorICD from the CheckMate™ Mammalian Two-Hybrid System. Luciferase expression was analyzed using the Bright-Glo™ Luciferase Assay System. (3376)

Notes: To determine the potential role that T-bet, a T-box transcription factor that specifies Th1 lineage commitment, and Hlx, a homeobox gene, may have in helper T cell differentiation, the physical interaction between the two proteins was tested using the CheckMate™ Mammalian Two-Hybrid System. The pACT-T-bet and pBIND-Hlx fusion vectors were co-transfected with pG5luc into 293T cells. After 48 hours, the firefly and Renilla luciferase activities were measured using the Dual-Glo^® Luciferase Assay System. (3493)

Notes: The authors of this study have developed a high throughput method for screening protein-protein interactions in 384-well plates. Using elements of the CheckMate™ Mammalian Two-Hybrid System, mouse cDNAs were linked to the GAL4 DNA binding and VP16 transcription activation domains via a linear cassette PCR strategy. The linear DNAs were mixed with the pG5luc plasmid, transfection reagent and CHO-K1 cells.  Twenty hours after transfection, activation of luciferase by GAL4 binding was assayed using the Steady-Glo^® Luciferase Assay System. Biotec and Tecan liquid handlers were used to semiautomate the system, allowing screening of 20,000 assay wells per day. (2727)

Notes: To study the transcriptional control of human CYP8B1 by bile acids, the -514/+300 region of the gene was cloned into the pGL3-Basic Vector and subsequently mutagenized at both the 5´ and 3´ends of the CYP8B1 fragment.  The various reporter constructs were then transfected into HepG2 cells and the activity assessed using the Luciferase Assay System. Luminescence was determined using a Berthold Lumat LB 9501 luminometer. The Gal4/Id and VP16/MyoD provided in the CheckMate™ Mammalian Two-Hybrid System were used as positive controls for the in a two-hybrid assay that examined the interaction of SHP and HNF4alpha, genes that regulate CYP8B1.  The TNT^® Quick Coupled Transcription/Translation System was used to synthesize various receptor proteins cloned in expression plasmids. (3083)

Notes: The pTARGET™ Mammalian Expression Vector was used to clone and express a portion of the ARA9, a protein that interacts with the aryl hydrocarbon receptor. The expressed protein was used in a two-hybrid assay using a GAL4-fusion protein containing a portion of the aryl hydrocarbon receptor. Interaction of the two proteins is reported by activation of the luciferase in the pG5-luc Vector, which is a portion of the CheckMate™ Mammalian Two-Hybrid System. The two-hybrid studies were performed in COS-1 cells and were controlled with a cotransfection with a β-galactosidase vector. Cells were lysed with the Passive Lysis Buffer and luciferase activities determined with the Luciferase Assay System. (0842)

Notes: The CheckMate™ Mammalian Two-Hybrid System was used as a one-hybrid system to measure transactivation activity. Various domains of the ATF6 protein were cloned in to the CheckMate™ Mammalian Two-Hybrid system vector pBIND, to determine the regions which contribute to transactivation of the pG5luc vector. The assays were done in HeLa cells. (2132)

Notes: The CheckMate™ Mammalian Two-Hybrid System was used to demonstrate a direct interaction between the transactivation domain of nuclear factor I-C (NFIC) and the CREB binding domain of the CREB-binding protein (CBP). Four different domains of the CBP were linked to the GAL4 binding domain of the pBIND Vector, and the transactivation region of the NFIC was cloned into the pACT Vector. The positive control vectors included with the system, pBIND-Id and pACT-myo Vectors, induced a 110-fold increase in luciferase activity. One construct of the CBP protein in combination with the NFIC construct induced a 70-fold increase in luciferase activity. Equal amounts of the vectors (0.6µg) were transfected into HepG2 cells. The Dual-Luciferase^® Reporter Assay System was used to assess luciferase activities. (0851)

Notes: The CheckMate™ Mammalian Two-Hybrid System was used to demonstrate an interaction between TIP49a and TIP49b in vivo. TIP49a or TIP49b were placed into the pBIND and pACT Vectors, and 50ng of each transformed into HeLa cells with 200ng of the pG5luc Vector. Both TIP49a and TIP49b were placed into the pBIND Vector and both were placed into the pACT Vector, so that all combinations of the constructs could be tested. The pBIND-TIP49b construct with the pACT-TIP49a construct produced a 15-fold increase in luciferase activity. The pBIND-TIP49a and pACT-TIP49b produced a 22-fold increase. TIP49b in both the pBIND and pACT Vectors produced only a 6-fold increase, and TIP49a in both vectors produced only a 2-fold increase. The increase in luciferase activity was in relation to the pBIND and pACT Vectors alone. The positive control vectors included in the system, pACT-MyoD Vector and the pBIND-Id Vector, produced a 221-fold increase. All firefly luciferase activities from the pG5luc Vector were normalized to the Renilla luciferase activity coming from the pBIND Vector, and both luciferase activities were determined with the Dual-Luciferase^® Reporter Assay System. (0958)