Requirement of E1B55K BC-box. (A) H1299 or H1299-HACul5 cells (lanes noted at bottom of the figure) were transfected with plasmid DNAs expressing E1B55K and/or E4orf6, as indicated at the top of the figure. Whole-cell lysates were either first immunoprecipitated (IP) (top two panels) using either 1807 anti-E4orf6 or anti-E1B55K antibodies as indicated on top of the figure or examined directly (bottom four panels) by Western blotting with antibodies against HA (for HA-Cul5), E1B55K, E4orf6, or elongin C (Elon C), as indicated in the figure. (B) H1299-HACul5 cells were transfected with combination of plasmid DNAs expressing wt or BC-box mutants of E1B55K (C184T) and E4orf6 (functional mutant 1, L47G), as indicated at the top of the figure. As in panel A, immunoprecipitates prepared using 1807 anti-E4orf6 antibodies or whole-cell extracts were examined by Western blotting using antibodies against HA (HA-Cul5), elongin C, E1B55K, or E4orf6, as indicated in the figure. (C) H1299 cells were transfected with the combination of plasmid DNAs expressing E4orf6 and the wt or BC-box mutant of E1B55K (C184T), as indicated at the top of the figure. Whole-cell lysates were analyzed for Mre11 expression levels by using anti-Mre11 antibodies.