Journal: Cell Reports
Article Title: HECTD3 Mediates an HSP90-Dependent Degradation Pathway for Protein Kinase Clients
doi: 10.1016/j.celrep.2017.05.078
Figure Lengend Snippet: The DOC Domain of HECTD3 Mediates Substrate Interaction (A) Schematic of the domain architecture of HECTD3. Only two defined domains can be recognized: the C-terminal catalytic HECT domain common to all members of the HECT E3 ubiquitin ligase family and a DOC domain homologous to APC10, which occurs midway through the N-terminal region. (B) Coomassie-stained SDS-PAGE of purified recombinant HECTD3 constructs: His 6 -tagged full-length HECTD3 and GST fusions of the isolated DOC and HECT domains. M, molecular weight, in kilodaltons. (C) Top: lysates from HEK293 cells expressing either eYFP or eYFP-CRAF were incubated with purified HECTD3 constructs as in (A), subjected to immunoprecipitation using GFP-Trap , and analyzed by western blot using α-His (left) or α-GST (right). Full-length HECTD3 and the isolated DOC domain, but not the isolated HECT domain, were co-immunoprecipitated from eYPF-CRAF cells. Bottom: loading control for above, western blotted with α-GFP. (D) Purified GST-DOC was added to HEK293 lysates treated with increasing concentrations of the HSP90 inhibitor AUY922 from 0, 100, 200, 400, 800, 1,600, and 3,200 nM and immunoprecipitated. Endogenous CRAF and HSP90 were co-immunoprecipitated, with increased yields at the higher drug concentrations.
Article Snippet: In these experiments, pretreated GST-Trap resin (Chromotek, sta-200) was used, and co-immunoprecipitations of CRAF and HSP90 were analyzed by western blotting.
Techniques: Staining, SDS Page, Purification, Recombinant, Construct, Isolation, Molecular Weight, Expressing, Incubation, Immunoprecipitation, Western Blot
