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J. Cell Sci. 127, 5261-72. Epiprofin orchestrates epidermal keratinocyte proliferation and differentiation. 2014

Nakamura, T., Yoshitomi, Y., Sakai, K., Patel, V., Fukumoto, S. and Yamada, Y.

Notes: Epiprofin (Epfn) was expressed in HaCaT cells from various HaloTag® Fusion vectors with either full-length or deletion mutants of the CMV promoter to provide different levels of Epfn expression. (The authors do not state whether the N-terminal or C-terminal HaloTag® Fusion vectors were used). HaCaT 72hr proliferation was highest when using the CMVd3 promoter (e.g., pFC17A or pFN24A) for the lowest expression level. Reporter assays were performed on in the HaCaT cells as well and were measured with the Dual-Luciferase® Reporter Assay System on a GloMax® Instrument. Transfection studies performed with primary keratinocytes utilized the ViaFect™ Transfection Reagent using the reverse transfection method. (4690)

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J. Biol. Chem. 287, 14052–68. MDM2 protein-mediated ubiquitination of NUMB protein: Identification of a second physiological substrate of MDM2 that employs a dual-site docking mechanism. 2012

Sczaniecka, M., Gladstone, K., Pettersson, S., McLaren, L., Huart, A.S. and Wallace, M.

Notes: The E3 ubiquitin ligase murine double minute 2 (MDM2) regulates transcriptional activity and expression levels of the p53 tumor suppressor protein. In this article, the authors show that the cell fate determinant NUMB interacts with MDM2, disrupting the interaction of MDM2 and p53 and preventing the ubiquitination of p53. To study protein-protein interactions between NUMB and MDM2, the authors used the pFN2A (GST) Flexi® Vector to express full-length and partial NUMB sequences as GST fusion proteins for protein pull-down experiments. To examine their hypothesis that the interaction between NUMB and MDM2 increases p53 levels and thus apoptosis in cells, they treated ZR75 human breast carcinoma cells and A375 human melanoma cells with NUMB-derived peptides and small molecule ligands that block the interaction between NUMB and MDM2. These treatments resulted in increased levels of p53 protein, Annexin V staining as well as caspase-3/7 activity, as determined using the Caspase-Glo® 3/7 Assay System. (4351)

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J. Biol. Chem. 283, 21579–87. ATP modulation of Ca2+ release by type-2 and type-3 inositol (1, 4, 5)-triphosphate receptors. Differing ATP sensitivities and molecular determinants of action. 2008

Betzenhauser, M.J., Wagner, L.E. 2nd, Iwai, M., Michikawa, T., Mikoshiba, K. and Yule, D.I.

Notes: The authors examined the different ATP sensitivities of inositol (1,4,5)-triphosphate receptor (InsP3R) isoforms InsP3R1, InsP3R2 and InsP3R3. To compare the ATP-binding properties of InsP3R2 and InsP3R3, nucleotide sequences encompassing the ATP-binding domains were amplified by PCR and cloned into the pFN2A (GST) Flexi® Vector. The ATP-binding sites were expressed as glutathione-S-transferase (GST) fusion proteins in BL21(DE3)pLysS cells. Fusion proteins were purified, and the GST tag removed by cleavage with tobacco etch virus (TEV) protease. Purified proteins were then used in a fluorescent ATP-binding assay. (3901)

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J. Biol. Chem. 281, 17410-17419. ATP binding to a unique site in the type-1 S2- inositol 1,4,5-triphosphate receptor defines susceptibility to phosphorylation by protein kinase A. 2006

Wagner, L.E., Betzenhauser, M.J. and Yule, D.I.

Notes: N-terminal GST fusion proteins of portions of the type-1 S2- Ionsitol 1,4,5-triphosphate receptor were created using the pFN2A (GST) Flexi® Vector. The constructs were expresed in BL21 (DE3) pLysS cells and used for ATP-binding assays. (3392)

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Hum. Mol. Genet. 15, R31-R43. From genome to proteome: developing expression clone resources for the human genome. 2006

Temple, G., Lamesch, P., Milstein, S., Hill, D.E., Wagner, L., Moore, T. and Vidal, M.

Notes: The Kazusa cDNA Project is constructing a library of more than 1,000 "full ORF" (F-ORF) clones in the Flexi® Vector System to characterize the function of proteins that are larger than 50kD. (3393)

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