Notes: These authors first used a FLAG-tagged protein (nfr2) with a HeLa Nuclear extract and captured interacting proteins via SDS-PAGE and in-gel digests of bands to identify (Krüppel-associated box)-associated protein 1 (KAP1) as a potential interacting partner. Human KAP1 was purchased as a HaloTag^® CMV Flexi^® Vector from Kazusa and used in a Mammalian PullDown scenario (with HaloLink™ Resin) to demonstrate interaction between the two proteins. A reporter assay was used to show that KAP1 facilitates Nrf2 transactivation in a dose-dependent manner. The authors defined the interaction sites using GST-tagged nrf2 and various forms of KAP1-HaloTag® Fusions expressed in TNT® SP6 High-Yield Wheat Germ Extract. GST-tagged proteins were expressed in E. coli and bound to glutathione-Sepharose beads. These bound proteins were mixed with the KAP1 from the cell-free expression system, incubated for 4 hours at 4°C, washed and stained with the HaloTag^® TMR Ligand for 30 minutes. The proteins from the pull-down assay were subjected to SDS-PAGE and the HaloTag^® proteins detected by phosphorimaging and the GST proteins by Coomassie Brilliant Blue Staining. A two-hybrid system consisting of the pRL-TK Vector with a firefly luciferase reporter with Gal4 UAS, mouse Nrf-2 N-terminal domain and KAP1 was also used. The vectors were transfected into Nrf2 knockout MEFs for 4 hours then incubated for 36 hours before luciferase expression was determined using the Dual-Luciferase^® Reporter Assay System. (4123)

Notes: These authors used the Flexi^® Vector System to prepare ORF clones encoding 1929 human genes and to transfer a subset of these clones to various expression vectors for further analysis. They created HaloTag^® fusion proteins and examined expression of these proteins in vitro and in COS7 and HEK293 cells. They also performed comparisons between the Flexi^® System and Gateway^® cloning system, specifically examining the effects of flanking sequences on protein expression in in vitro translation systems and confirming that the cellular localization of the HaloTag^® fusion proteins was consistent with results obtained using GFP-fusions. (3800)

Notes: RBCK1 is a RING-IBR (ring in between ring fingers) protein previously shown to have transcriptional activity and to bind protein Kinase Cβ. This paper demonstrates that RBCK1 also possesses ubiquitin ligase E3 activity. Both FLAG-tag and HaloTag® labeled proteins were used to demonstrate this activity. To demonstrate the interaction between RBCK1 and ubiquitinated proteins, HaloTag®-ubiquitin and FLAG-RBCK1 were coexpressed in HEK293 cells in the presence or absence of a proteasome inhibitor. The anti-FLAG immunoprecipitates isolated from these cells were analyzed by SDS-PAGE and Western blotting using anti-HaloTag® and anti-FLAG antibodies and self-ubiquitination of RBCK1 was demonstrated. RBCK2, a splice variant of RBCK1 lacking the RING domain showed no self ubiquitination activity but was demonstrated to interact with RBCK1 in vivo and in vitro, and to inhibit the self-ubiquitination activity of RBCK1 in a FLAG-tag assay. Pulse-chase experiments, using HEK293 cells expressing HaloTag®-RBCK1 with or without RBCK2 and treated with HaloTag®-TMR ligand, were used to show that the half-life of RBCK1 was extended by overexpression of RBCK2. (3874)