Fig. 7.

N-terminal phosphorylated Cdh1 is translocated to the cytoplasm by CRM1. a Immunofluorescent staining of shCdh1-MDA-MB-231 cells stably expressing WT-, 4D-, 4A-, Y148E-, or Y148F-Cdh1 using anti-HA(Cdh1) antibody, DAPI was used for DNA staining. *Cdh1 cDNA used in this experiment has been mutated to escape shCdh1-mediated gene silencing. Scale bar, 50 μm. b MDA-MB-231 cells generated in (a) were subjected to cytoplasm/nucleus fractionation followed by immunoblot (IB) analysis. c shCdh1-MDA-MB-231 cells stably expressing the indicated Cdh1 retroviral constructs were subjected to cytoplasm/nucleus fractionation followed by IB analysis. d IB analysis of whole-cell lysates (WCL) and anti-Flag immunoprecipitates (IP) derived from 293T cells transfected with Flag-CRM1 and the indicated HA-Cdh1 constructs. e IB analysis of WCL and anti-HA IP derived from MDA-MB-231 cells generated in (a). f MCF7 and MDA-MB-231 cells were infected with shScr or shCRM1 lentiviral shRNA constructs. The infected cells were selected with 1 μg ml⁻¹ puromycin for 72 h before harvest for cytoplasm/nucleus fractionation and IB analysis. g shCdh1-MDA-MB-231 cells stably expressing WT-, 4D-, Y148E-Cdh1 were infected with shScr or shCRM1 lentiviral shRNA constructs. The infected cells were selected with 1 μg ml⁻¹ puromycin for 72 h before harvest for cytoplasm/nucleus fractionation and IB analysis. h MDA-MB-231 cells were subjected to cytoplasm/nucleus fractionation followed by anti-Cdh1 IP and IB analyses