Figure 3.

Monoubiquitination of AMAP1 by Cbl. (A) Ubiquitination of AMAP1. HEK293T cells were transfected with AMAP1-FLAG, Xpress-CIN85, Cbl-b and HA-ubiquitin, as indicated. FLAG-tagged proteins were then immunoprecipitated from 200 μg of cell lysates using an anti-FLAG antibody and subjected to blotting with an anti-HA antibody (at 1:20 000 dilution). (B) Requirement of Cbl for AMAP1 ubiquitination. HEK293T cells, treated with siRNA duplexes for c-Cbl and Cbl-b, or with an irrelevant sequence (irr) were transfected with AMAP1-FLAG and HA-ubiquitin. FLAG-tagged proteins were then immunoprecipitated from 200 μg of cell lysates and subjected to blotting with an anti-HA antibody (at 1:1000 dilution). (C) Evidence for the monoubiquitination of AMAP1. HEK293T cells were transfected with GST-AMAP1, Xpress-CIN85, Cbl-b and HA-ubiquitin. GST-fusion proteins were then pulled down from 200 μg of cell lysates and subjected to immunoblotting, as indicated. (D) Effects of proteasome inhibitors on AMAP1 and its ubiquitination. HEK293T cells, transfected with GST-AMAP1 and HA-ubiquitin, were treated with MG132 (20 μM), Epoxomicin (2 μM), Lactacystin (10 μg/ml), ZLLH (20 μM) or E64 (100 μg/ml) for 5 h. GST-AMAP1 was then pulled down from 200 μg of cell lysates and subjected to immunoblotting with an anti-HA antibody. (E) Effects of proteasome inhibitors on endogenous AMAP1 in MDA-MB-231 cells. Cells were treated with proteasome inhibitors or related compounds, as above. Total cell lysates were then subjected to immunoblotting, as indicated. (F) Ubiquitination of AMAP1 in MDA-MB-231 cells. Cells were transfected with AMAP1-FLAG and HA-ubiquitin. AMAP1-FLAG was then immunoprecipitated from 500 μg of cell lysates using an anti-FLAG antibody and subjected to immunoblotting with an anti-HA antibody. In (A–F), data are representative of more than three experiments. Total, 10 μg of total cell lysates. In (A–C), AMAP2 was included as a control.