vFLIP and RTA interact with endogenous Itch. A. RTA interacts with endogenous Itch and vFLIP. 293T cells were transfected with vFLIP and RTA. 24h post-transfection, cells were treated with 1uM MG132. 48h post transfection, cells were harvested and processed for immunoprecipitation. Precleared lysates were incubated with antibodies against RTA followed by addition of protein A/G agarose and washed with RIPA buffer. Immunoprecipitates were analyzed by SDS-PAGE followed by immunoblot using antibodies against myc, RTA, and Itch. B. vFLIP interacts with endogenous Itch. 293T cells were transfected with vFLIP and RTA. 24h post-transfection, cells were treated with 1uM MG132. 48h post transfection, cells were harvested and processed for immunoprecipitation. Precleared lysates were incubated with antibodies against myc-vFLIP followed by addition of protein A/G agarose and washed with RIPA buffer. Immunoprecipitates were analyzed by SDS-PAGE followed by immunoblot using antibodies against myc and Itch. *Asterisk denotes light chain (LC) band.