Fig. 1.

Cdh1 negatively regulates Src kinase activity in an APC-independent manner. a–c Immunoblot (IB) analysis of MCF7 (a), MDA-MB-231 (b), and BT474 (c) cells infected with control (shScramble, shScr for short) or the indicated shCdh1 lentiviral shRNA constructs. The infected cells were selected with 1 μg ml⁻¹ puromycin for 72 h before harvest. d CRISPR/Cas9-mediated deletion of Cdh1 activated Src. IB analysis of MCF7 cells infected with control (sgGFP) or sgCdh1 lentiviral construct. The infected cells were selected with 1 μg ml⁻¹ puromycin for 7 days before plating for single clone selection. e Src was activated in Cdh1^−/− MEFs. IB analysis of WT and Cdh1^−/− MEFs treated with 4 ng ml⁻¹ PDGF for the indicated periods of time after 16 h serum deprivation. f IB analysis of MCF7 cells infected with the indicated lentiviral shRNA constructs. The infected cells were selected with 1 μg ml⁻¹ puromycin for 72 h before harvest. g, h IB analysis of MCF7 cells infected with control (shScr) or the indicated shCdc27 (g) and shAPC10 (h) lentiviral shRNA constructs. The infected cells were selected with 1 μg ml⁻¹ puromycin for 72 h before harvest. i MCF7 cells stably expressing retroviral empty vector (EV), WT-, or N-Cdh1 were further infected with shScr or shCdh1 lentiviral constructs as indicated. The infected cells were selected with 1 μg ml⁻¹ puromycin for 72 h before harvest. *Cdh1 cDNA used in this experiment has been mutated to escape shCdh1-mediated gene silencing. ** indicates nonspecific bands. j IB analysis of T47D cells infected with the indicated lentiviral shRNA constructs. The infected cells were selected with 1 μg ml⁻¹ puromycin and 100 μg ml⁻¹ hygromycin for 72 h before harvest. k IB analysis of WCL derived from sgGFP- and sgCdh1-infected MDA-MB-231 cells that were synchronized at the G1–S boundary by double-thymidine block and then released back into the cell cycle for the indicated periods of time