(A–B) Co-immunoprecipitation of p27 and Cortactin: (A) p27 was immunoprecipitated using rabbit anti-p27 (C19) antibodies from HEK293 lysates transfected with plasmids encoding p27, Myc-Cortactin (Myc-Cort) or both. (B) Immunoprecipitation of endogenous Cortactin using rabbit anti-Cortactin (H191) antibodies from HeLa or E6 MEF lysates, beads alone were used as control. (A–B) Co-immunoprecipitated proteins were detected by immunoblot with mouse anti-c-Myc (9E10) (A) or mouse anti-p27 (F-8) antibodies (B). Immunoprecipitated proteins were visualized by reprobing the membrane with mouse anti-p27 (F-8) (A) or with rabbit anti-Cortactin (H-191) antibodies and anti-rabbit Ig light-chain secondary antibodies (B). Immunoblots of extracts show the level of proteins in each condition. (C–F) Pull-down assays: HEK293 cells were transfected with Myc-Cortactin (C–E) or various deletion mutants of Myc-Cortactin (F) (ΔABR6, ΔABR5-6, ΔABR4-6, ΔABR3-6, ΔABR, ΔABR/NTA or ΔSH3, described in the schematic representation of Cortactin, bottom panel). NTA: N-Terminal acidic domain; ABR: actin binding repeat; Helix: helical domain; SH3: Src-homology three domain. Lysates were subjected to pull-down assays using GST, GST-p27 or GST-p27CK- (C), or GST, GST-p27, GST-p27 NT (1-87) and GST-p27 CT (88-198) (D), or GST, GST-p27CK-, GST-p27CK- (1-197) and GST-p27CK- (1-190) (E) or GST and GST-p27 (F). The amounts of Myc-Cortactin bound to the beads and of transfected protein present in the extracts were detected by immunoblot using mouse anti c-Myc (9E10) antibodies. The amounts of GST or GST p27 and mutants used in the assays were visualized by Coomassie staining of the gels. (A–F) All panels show representative results of at least three independent experiments.