Figure 6.

SCP4 physically interacts with Snail. (a) Endogenous SCP4 and Snail were co-immunoprecipitated in MCF10A cells. MCF10A cells stably expressing HA-Snail were harvested, and the lysates were immunoprecipitated with anti-HA antibody or control IgG. The co-IP complexes and the inputs were analysed by western blotting with the indicated antibodies. (b) SCP4 interacts with Snail in vivo. HEK293T cells were co-transfected with HA-Snail and FLAG–SCP4 or FLAG–SCP4DN. SCP4 was immunoprecipitated (IP) with anti-FLAG antibody and then subjected to SDS–PAGE and western blotting (IB) to detect SCP4 bounded Snail. WCL, whole cell lysate. (c) SCP4 interacts with Snail in vivo. HEK293T cells were co-transfected with HA-Snail and FLAG–SCP4 or FLAG–SCP4DN. Snail was immunoprecipitated (IP) with anti-HA antibody and then subjected to SDS–PAGE and western blotting (IB) to detect Snail-bounded SCP4. (d) Direct interaction between SCP4 and Snail in vitro. GST-SCP4 fusion protein was expressed in E. coli and purified by glutathione beads. In vitro translated HA-Snail was incubated with purified glutathione bead-bound GST protein or GST-SCP4. The retrieved complex was subjected to SDS–PAGE and western blotting analysis. (e) SCP4 interacts with the N-terminal region of Snail. HEK293T cells were co-transfected with FLAG–SCP4 and HA-Snail-WT or its deletion mutant HA-Snail-1-153, HA-Snail-153-264. Snail was immunoprecipitated (IP) with anti-HA antibody and then subjected to SDS–PAGE and western blotting (IB) to detect Snail bounded SCP4. (f) SCP4 directly interacts with the N-terminal region of Snail in vitro. Full length GST-Snail fusion protein or its deletion mutant GST-Snail-1-153 or GST-Snail-153-264 were expressed in E. coli and purified by glutathione beads. In vitro translated FLAG–SCP4 was incubated with purified glutathione bead-bound GST protein, GST-Snail, GST-Snail-1-153 or GST-Snail-153-264. The retrieved complex was subjected to SDS–PAGE and western blotting analysis.