Figure 4.
MOAP-1 is an APC/CCdh1 substrate with D-box motifs. (A) Sequences of putative D-boxes on MOAP-1. D-box consensus amino acids mutated to Ala and transfected MOAP-1 WT or D-box mutant constructs into 293T cells are underlined. (B) 293T cells were transfected with HA-Cdh1 and Myc–MOAP-1 WT or MOAP-1 MT. Cells were collected and immunoprecipitated (IP) by anti-HA antibody. Samples resolved by SDS-PAGE were immunoblotted using anti-Myc or -HA antibodies. (C) MBP or MBP–MOAP-1 WT or MBP–MOAP-1 MT was incubated with His-Cdh1 for 2 h at 4°C, and proteins were retrieved from the mixture using amylose beads. After SDS-PAGE, samples were immunoblotted with anti-MBP or -Cdh1 antibodies. (D) 293T cells were transfected with Myc–MOAP-1 WT or Myc–MOAP-1 MT and treated with 100 µM cycloheximide (CHX). Cell lysates were prepared at the indicated times after cycloheximide treatment and immunoblotted with anti-Myc or -actin antibodies. (E) 293T cells were transfected with Myc–MOAP-1 WT or MT in the presence or absence of HA-Cdh1. After 48 h, cells were treated with 20 µM MG132 for 16 h, and lysates were collected for immunoblotting with anti-Myc, -HA, or -actin antibodies. (F) Recombinant MBP–MOAP-1 WT or MT proteins were incubated with ubiquitin, E1 and E2 (UbcH10 and Ube2S), ATP, and APC/C precipitated from HeLa G1 cell lysates using anti-Cdc27 antibody. Samples were immunoblotted with anti–MOAP-1 antibody.