Figure 2

Cdc20 interacts with and promotes proteasomal degradation of SMAR1. (a) Cdc20 degrades SMAR1 through proteasome. MCF7 cells were transfected with either vector or HA-Cdc20 for 36 h and then treated with or without 10 μM MG132 for 6 h. Cells were then harvested, prepared protein extracts and immunoblotted for indicated antibodies. (b) Cdc20 targets both cytoplasmic and nuclear fraction of SMAR1. MCF7 cells were transfected with either vector or HA-Cdc20 for 48 h. Cells were then collected and fractionated for cytoplasmic and nuclear pool followed by immunoblotting for indicated antibodies. (c) Cdc20 interacts with SMAR1. MCF7 cells were transfected either vector or combination of HA-Cdc20 and FLAG-SMAR1 for 36 h. Transfected cells were then treated with 10 μM MG132 for 6 h. Whole cell protein extracts were used for immunoprecipitation (IP) with indicated antibody. The immune precipitates were then immunoblotted for indicated proteins. (d) SMAR1-Cdc20 interacts at the endogenous level. MCF7 cell extracts were immunoprecipitated with either IgG or SMAR1 antibody followed by immunoblotted for indicated proteins. (e) Cdc20 promotes polyubiquitylation of SMAR1 in vivo. MCF7 cells were co-transfected with indicated plasmids for 36 h.Transfected cells were then treated with 10 μM MG132 for last 6 h. Whole cell protein extracts were immunoprecipitated with anti-FLAG antibody and immunoprecipitates were immunoblottedfor ubiquitin. (f) Cycloheximide pulse chase assay showed the differential turnover kinetics of SMAR1 in MCF7 and MDA-MB-231 cell lines. (g) Comparative polyubiquitination profile of SMAR1 in MCF7 and MDA-MB-231. MCF7 and MDA-MB-231 cells were treated with 10 μM MG132 for 6 h and lysates were immunoprecipitated for SMAR1 and the immunoprecipitates were probed for anti-K48-linked ubiquitin