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)

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)

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)

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)

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)