FIG 1.

Eya1 is posttranslationally modified by ubiquitin. (A and B) A proteasome inhibitor, MG132, increases the Eya1 protein level. HEK293 cells that were stably transfected with a Flag-Eya1 expression vector were treated with MG132 (12.5 μM). The Flag/Eya1 protein level in total cell lysates was determined using a Flag-specific antibody after 16 h of MG132 treatment (A) or during a time course (B). GAPDH was used as a protein loading control. DMSO mock treatment was considered the starting point (0 h). (C and D) Eya1 is ubiquitinated in HEK293 cells. Flag/Eya1, Flag/D327A, and HA-tagged ubiquitin (HA/UB) were transiently coexpressed in HEK293 cells. An immunoprecipitation/immunoblotting (IP/IB) assay was done using the indicated antibodies or control IgG at 48 h after transfection. Numbers on the side of each panel indicate the ubiquitin chain length.