Figure 1. Cdh1 interacts with Smurf1 to promote Smurf1 auto-ubiquitination.
A. Immunoblot (IB) analysis of 293T whole cell lysates (WCL) and anti-Cdh1 immunoprecipitates (IP). Mouse IgG was used as a negative control for the immunoprecipitation procedure.
B. Autoradiography of 35S-labelled Smurf1 bound to the indicated GST fusion proteins.
C. Immunoblot (IB) analysis of whole cell lysates (WCL) and immunoprecipitates (IP) derived from 293T cells transfected with HA-Cdh1 or HA-Cdc20 and Flag-Smurf1 constructs. Thirty hours post-transfection, cells were pretreated with 10 μM MG132 for 10 hours to block the proteasome pathway before harvesting.
D. Immunoblot (IB) analysis of whole cell lysates (WCL) derived from 293T cells transfected with HA-Cdh1 or HA-Cdc20 together with Myc-tagged Smurf1 constructs. Where indicated, 10 μM MG132 was added for 10 hours to block the proteasome pathway before harvesting.
E. Immunoblot (IB) analysis of whole cell lysates (WCL) derived from 293T cells transfected with HA-Cdh1 and the indicated Myc-tagged Smurf1 or Smurf2 constructs. CA-Smurf1 is an E3 ligase activity-deficient mutant form of Smurf1 in which the cysteine residue in the active site is replaced with alanine.
F. Immunoblot (IB) analysis of whole cell lysates (WCL) derived from 293T cells transfected with Myc-tagged Smurf1 or CA-Smurf1 in the presence of increasing amounts of HA-Cdh1. Where indicated, 10 μM MG132 was added for 10 hours to block the proteasome pathway before harvesting.
G. Schematic representation of the Cdh1 protein functional domains.
H. Immunoblot (IB) analysis of whole cell lysates (WCL) derived from 293T cells transfected with Myc-Smurf1 and the indicated HA-Cdh1 constructs.
I. Immunoblot (IB) analysis of whole cell lysates (WCL) derived from HeLa cells transfected with Flag-Cyclin B1 and the indicated HA-Cdh1 constructs.
J. Immunoblot analysis (IB) of whole cell lysates (WCL) and anti-Flag immunoprecipitates (IP) derived from 293T cells transfected with the indicated plasmids. Twenty hours post-transfection, cells were treated with the proteasome inhibitor MG132 overnight before harvesting.
K. Immunoblot analysis (IB) of whole cell lysates (WCL) and anti-Smurf1 immunoprecipitates (IP) derived from HCT116 cells infected with the indicated lentiviral shRNA constructs. Twenty hours post-infection, cells were selected with 1 μg/ml puromycin for 72 hours to eliminate non-infected cells. Cells were treated with the proteasome inhibitor MG132 overnight before harvesting.
L. Purified recombinant GST-Cdh1 augments Smurf1 auto-ubiquitination in vitro. Bacterially expressed and purified GST-Smurf1 was incubated with the indicated, purified recombinant GST-Cdh1 or GST-CKIP1 proteins, E1, E2 and ubiquitin as indicated at 30° C for 15 minutes. The ubiquitination reaction products were resolved by SDS-PAGE and probed with the indicated antibodies.
(see also Figure S1)