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J. Biol. Chem. 281, 9030-9037. The plasma membrane lactate transporter MCT4, but not MCT1, is up-regulated by hypoxia through a HIF-1alpha-dependent mechanism. 2006

Ullah, M.S., Davies, A.J. and Halestrap, A.P.

Notes: Monocarboxylate transporters (MCT) transport lactic acid across the cell membrane. The promoters of 4 MCT family members (MCT1, MCT2, MCT3 and MCT4), were amplified by PCR and cloned into the pGEM®-T Easy Vector. The sequences were confirmed, and the promoters were cloned into the pGL3-Basic Vector. The Dual-Glo™ Luciferase Assay System was used to quantitate promoter activity under basal and hypoxic conditions in HeLa cells. The pRL-SV40 Vector was used to normalize for differences in transfection efficiency. (3463)

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J. Immunol. 175, 5981–5985. Transcriptional activators of helper T cell fate are required for establishment but not maintenance of signature cytokine expression. 2005

Martins, G.A., Hutchins, A.S. and Reiner, S.L.

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)

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J. Immunol. 172, 5512–5521. Human CD1D gene has TATA boxless dual promoters: An SP1-binding element determines the function of the proximal promoter. 2004

Chen, Q.Y. and Jackson, N.

Notes: These authors demonstrated that the human CD1D gene has distal and proximal TATA boxless promoter sequences. Distal and proximal promoters to CD1D were cloned into the pGL3-Basic Vector to create reporter constructs. One construct contained the entire 4,986 base pair region containing distal and proximal CD1D promoter.  Transient transfections were performed using 5 x 105 Jurkat cells in 24-well plates, 0.8 μg of pGL3 Basic Vector with the insert of interest, and 30ng of pRL-CMV Vector as a transfection normalization control. The Dual-Glo™ Luciferase Assay System was used to assay luciferase activities.  (3061)

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RNA 10, 277–286. Localization of a promoter in the putative internal ribosome entry site of the Saccharomyces cerevisiae TIF4631 gene. 2004

Verge´, V., VonLanthenm, M., Masson, J.M., Trachsel, H., and Altmann, M.

Notes: Researchers cloned the Photinus and Renilla luciferase ORFs into the pSP64 Poly(A) Vector to create a dual-reporter vector named SP6P. A similar vector, SP6R.4G(-508/-3).P, was created in which a 5´ untranslated region from the Saccharomyces cerevisiae TIF4631 gene was cloned between the two reporter genes. These two vectors were used to transform yeast strains. The resultant transformants were lysed using Passive Lysis Buffer and a modified lysis procedure.   Lysates were analyzed for luciferase activities using the Dual-Luciferase® Reporter Assay System and a TD20/20 luminometer. The researchers also cloned and sequenced the 5´  untranslated region of TIF4631 by using a RACE-PCR technique followed by cloning the PCR amplimers into the pGEM®-T Vector. (2845)

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Hum. Mol. Genet. 13, 2221-2231. SNPs in the promoter of a B cell-specific antisense transcript, SAS-ZFAT, determine susceptibility to autoimmune thyroid disease. 2004

Shirasawa, S., Harada, H., Furugaki, K., Akamizu, T., Ishikawa, N., Ito, K., Ito, K., Tamai, H., Kuma, K., Kubota, S., Hiratani, H., Tsuchiya, T., Baba, I., Ishikawa, M., Tanaka, M., Sakai, K., Aoki, M., Yamamoto, K. and Sasazuki, T.

Notes: Real-time TaqMan® amplification reactions were cleaned up using the Wizard® MagneSil® Sequencing Reaction Clean-Up System.  The purified products were then used in sequencing reactions.  This paper also describes use of the Dual-Luciferase® Reporter Assay System to analyze HEK293 cells transfected with a pGL3-Enhancer vector construct.  (3181)

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J. Virol. 77, 4588-4596. Human Cytomegalovirus activates inflammatory cytokine responses via CD14 and Toll-like Receptor 2. 2003

Compton, T., Kurt-Jones, E.A., Boehme, K.W., Belko, J., Latz, E., Golenbock, D.T., and Finberg, R.W.

Notes: Researchers used Promega's pGL3-Basic Vector to create a NF-κB promoter-driven firefly luciferase reporter vector for use in transient transfections with the phRL-TK Vector. The two vectors, along with pUC18, were transiently transfected into HEK293 cells using GeneJuice Transfection Reagent (Novagen). Transfections were performed overnight using 80ng NF-κB-pGL3-Basic construct, and 20ng of phRL-TK and pUC18 vectors in 96-well plates containing 2.5 x 104 cells/well. The following day, the cells were exposed to various treatments. Luciferase activity was measured using the Dual-Glo™ Luciferase Assay System. (2672)

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Mol. Cell. Biol. 22, 5182-5193. Drosophila Mi-2 Negatively Regulates dDREF by Inhibiting Its DNA-Binding Activity. 2002

Hirose, F., Ohshima, N., Kwon, E.-J., Yoshida, H., and Yamaguchi, M.

Notes: Drosophila Mi-2 was discovered to specifically bind the DNA binding domain of dDREF (DNA replication-related element factor) by yeast two hybrid screen. This binding was shown to inhibit dDREF’s transcriptional regulatory action on a DNA replication-related element (DRE). Analysis of dDREF binding to the DRE was performed by examination of expression from –168DPCNAluc, –168mutΔPCNAluc, and pRL-actin5C reporter genes in Schneider cells using the Dual-Glo™ Luciferase Assay System. (2622)

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