Figure 7.

Cdh1 regulates the Rb/E2F1 pathway. (A) Immunoblot analysis of primary human lung fibroblasts and primary human foreskin fibroblasts infected with indicated shRNA lentiviral vectors. Twenty-four hours after infection, cells were selected with 1 μg/ml puromycin to eliminate the noninfected cells. Whole-cell lysates were harvested 6 d after puromycin selection. (B) Immunoblot analysis of the U2OS cells that were engineered to overexpress Myc-Cdh1 after removal of tetracycline with indicated antibodies. (C–E) Autoradiograms showing recovery of ³⁵S-labeled Rb protein bound to GST-Cdh1 fusion proteins. Where indicated, in vitro–translated Rb protein was incubated with phosphatase (C) or cyclinA/Cdk2 kinase (D) before the pulldown assays. (E) Increased amount of in vitro–translated E2F1 protein was added to the binding reactions (3, 9, and 27 μl, as indicated by the triangles). (F) Autoradiograms showing recovery of ³⁵S-labeled Rb protein bound to GST-E2F1 fusion proteins. Where indicated, increased amount of in vitro-translated Cdh1 protein was added to the binding reactions (3, 9, and 27 μl, as indicated by the triangles). (G) Illustration of the proposed model by which Cdh1 could potentially regulate the abundance of E2F1 through the Rb protein.