Notes: The pCI Mammalian Expression Vector was used to express a flag-tagged 497 amino acid potassium channel in COS cells. (0433)

Notes: The 676 amino acid KvLQT1 protein and site-directed mutants were cloned into the pCI Mammalian Expression Vector, expressed in COS cells and assayed for activity. (1297)

Notes: The pCI Mammalian Expression Vector was used to express the p64H1 protein in 293 cells and HT-4, a clonal neuronal cell line. Inserts in the vectors were verified for expression using the TNT^® Coupled Reticulocyte Lysate System. The proteins were translated in the presence of Canine Pancreatic Microsomal Membranes (CMMs). The pGEM^®-T Vector System was used to clone the PCR product of circle rapid amplification of cDNA ends to find the true 5'-end of the p64H1 cDNA. The Tfx™-20 Reagent was used to transfect 293 cells and HT-4 neuronal cells with the p64H1-expressing pCI-based plasmid. (1206)

Notes: Single-stranded 3´-end biotinylated oligonucleotides were captured on the Streptavidin MagneSphere^® Paramagnetic Particles (SA-PMPs) and incubated with heparin-agarose affinity-purified nuclear proteins. The reaction was performed under the conditions used for electrophoretic gel shifts. Proteins were eluted in 1X nonreducing SDS-PAGE sample buffer at 65°C for 3 minutes and analyzed by Southwestern blotting with ³²P-labeled oligonucleotides. The ssDNA-affinity capture worked with the oligonucleotides prebound to the particles or after allowing the interactions to occur in solution prior to capture. The pCI Mammalian Expression Vector was used to express the proteins Purα (321 amino acids) and Purβ (324 amino acids) in mouse embryo-derived AKR-2B fibroblasts. Cell extracts were prepared and assayed by Southwestern blotting. The same proteins were in vitro translated with the TNT^® Coupled Reticulocyte Lysate System and assayed as well. (0929)

Notes: Single-stranded 3'-end biotinylated oligonucleotides were captured on the paramagnetic particles and incubated with heparin-agarose affinity-purified nuclear proteins. The reaction was performed under the conditions used for electrophoretic gel shifts. Proteins were eluted in 1X non-reducing SDS-PAGE sample buffer at 65°C for 3 min and analyzed by Southwestern blotting with 32P-labeled oligonucleotides. The ssDNA-affinity capture apparently worked with the oligonucleotides prebound to the particles or after allowing the interactions to occur in solution prior to capture. (1666)

Notes: The TASK protein cDNA was subcloned into the pCI Mammalian Expression Vector and expressed in COS cells. The potassium channel formed by this 395 amino acid protein was assayed by patch-clamp. (1205)

Notes: Both pCI and pCI-neo Mammalian Expression Vectors were used to express proteins in Mouse LTA and NIH3T3 cells and human HeLa cells. The authors report, 'In general we observed that even after optimization of the transfection conditions the expression level of the pCI-neo constructs was only 25% compared with pCI constructs.' (1122)

Notes: Both ZEB and the DNA-binding domain of ZEB were subcloned into Promega's pCI-neo Mammalian Expression Vector just behind a FLAG epitope. After cotransfection with reporter vectors, the effect of the expressed protein was assessed in 10T1/2 fibroblasts and C2C12 cells. The two protein inserts were subcloned into a T3 RNA polymerase binding site bearing plasmid and the flag-tagged proteins were expressed in vitro with the TNT^® Coupled Reticulocyte Lysate System. (0542)