Skip to main content
. Author manuscript; available in PMC: 2018 Jan 15.
Published in final edited form as: Virology. 2016 Nov 30;501:119–126. doi: 10.1016/j.virol.2016.11.016

Figure 2.

Figure 2

Itch ubiquitinates vFLIP. A. Itch ubiquitinates vFLIP in vitro. In 30ul reactions, the following reagents were added where indicated: Itch (0.5ug), Ub aldehyde (5 ug), fraction B (E1+E2) 12ul, 10X energy solution (3ul), vFLIP (5.5ug). Reactions were incubated at 37°C for 1.5h and stopped by addition of 2x Laemmli loading buffer. Reactions were analyzed by SDS PAGE followed by detection of vFLIP:ubiquitin conjugates with antibody against V5-vFLIP. B. Itch is required for RTA induced ubiquitination of vFLIP. 293T cells were transfected with HA tagged ubiquitin, myc tagged vFLIP, RTA, and Flag tagged Itch C830A where indicated. Cells were treated with MG132 4h prior to harvesting. Cell lysates were harvested for immunoprecipitation with anti HA. Immunoprecipitates were washed with RIPA lysis buffer containing 500mM NaCl and analyzed via immunoblot against HA, myc, FLAG, and RTA. (HC = heavy chain, LC = light chain)