A, A549 cells were treated with 1 μg/mL doxorubicin overnight. Before harvesting, cells were treated with MG132. Equal amounts of cell lysates were immunoprecipitated with either rabbit IgG (as control) or anti-COP1, and immunoblotted with anti-14-3-3σ (upper panel). Lysates were also immunoprecipitated with the indicated antibodies and immunoblotted with anti-COP1 (bottom panel). WCL: whole cell lysate.
B, 35S-labeled COP1 proteins were translated in vitro and incubated with either GST- or GST-14-3-3σ for binding assay. The bottom panel shows the input of GST proteins analyzed by Coomassie Blue staining. PD: pull down.
C, Bacterially purified Flag-COP1 domains were incubated with GST-14-3-3σ, and then was subjected to GST pull-down (PD) followed by immunoblotting using anti-Flag. Specific interaction of COP1 domains with 14-3-3σ is indicated by arrows. The asterisks indicate non-specific bands.