Figure 2. Post-translational modification of AIB1 at mitosis does not correlate with ubiquitination.

(A) HeLa cells constitutively expressing 6xhistidine tagged-ubiquitin were grown asynchronously or arrested at mitosis with nocodazole and cell lysates were either directly analyzed (total extract) or subjected to purification through a Ni²⁺-agarose column and further analyzed by western blotting. Arrow indicates the AIB1 mitotic-specific band. (B) MCF-7 cell extracts (30 µg, 1∶100) from arrested cells with nocodazole were subjected to enzymatic assay with the 300 ng of deubiquitinating enzyme GST-USP2 in 50 µl deubiquitylation buffer (50 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 mM DTT). Samples were analyzed by western blot with anti-mono, poly-ubiquitin antibodies and anti-AIB1 antibodies. The altered electrophoretic mobility of the AIB1 band is indicated with an arrow, demonstrating insensitivity to USP2 activity. (C) HeLa cells were grown asynchronously, or treated with the proteasome inhibitor MG132, or with etoposide to arrest them at G2 or with nocodazole to arrest them at mitosis (M). Cell lysates were immunoprecipitated with AIB1 antibodies and immunocomplexes were analyzed by western blotting with anti-mono, poly-ubiquitin antibodies and anti-AIB1 antibodies. The arrow indicates the mitosis-specific AIB1 band that is not detected with ubiquitin antibodies.