A, 293T cells were cotransfected with the indicated plasmids and increasing amounts of Flag-14-3-3σ. The cells were treated with MG132 for 6 hr before harvesting, and the ubiquitinated COP1 was immunoprecipitated with anti-Myc and immunobltted with anti-HA. Equal amounts of whole cell lysate (WCL) were immunoblotted with the indicated antibodies.
B, Indicated cells were treated with MG132 for 6 hr before harvesting. Polyubiquitinated COP1 was detected by immunoprecipitating with anti-COP1, followed by immunoblotting with anti-ubiquitin.
C, HCT116 cells were knocked down with two specific 14-3-3σ shRNA (1 & 2) or control Luciferase shRNA (C). Lysates were immunoprecipitated with anti-COP1 and immunoblotted with anti-ubiquitin.
D, Flag-COP1 and Flag-COP1 RING mutant (C136S, C139S) proteins were made using an in vitro Transcription/Translation system (TNT) and then incubated with the indicated purified GST proteins. Polyubiquitinated COP1 was examined by immunoprecipitating with anti-Flag, followed by immunoblotting with anti-ubiquitin (middle panel). The bottom panel shows the input of GST proteins (Coomassie Blue staining).