FIGURE 1:

The YOD1 deubiquitinase binds to the Hrd1 E3 ubiquitin ligase. (A) 293T cells were transfected with WT Hrd1-Myc alone or with FLAG-WT YOD1 and lysed in a buffer containing 1% NP-40. The resulting WCLs were incubated with FLAG antibody–conjugated beads, and the immunoprecipitates were subjected to SDS–PAGE followed by immunoblotting with the indicated antibodies. The corresponding WCLs were analyzed by SDS–PAGE and immunoblotting with the indicated antibodies. (B) Cells were transfected with FLAG-WT YOD1 and either WT Hrd1-Myc, cyt Hrd1-Myc, or TM1-6 Hrd1-Myc, and processed as in (A). *, An unidentified band that cross-reacts with the Myc antibody. (C) Cells were transfected with FLAG-WT YOD1 and WT Hrd1-Myc, C291A Hrd1-Myc alone, or C291A Hrd1-Myc and FLAG-WT YOD1, and processed as in (A). (D) Cells were transfected with WT Hrd1-Myc alone or with FLAG-WT YOD1 or FLAG-WT Atx3, and processed as in (A). (E) Cells were transfected with WT Hrd1-Myc alone, WT Hrd1-Myc and FLAG-WT YOD1, WT gp78-Myc alone, or WT gp78-Myc and FLAG-WT YOD1, and processed as in (A). At least three independent experiments were performed in each condition.