(A) Co-immunoprecipitation of endogenous PLK1 and CRAF from HeLa cell lysates obtained under control conditions or following nocodazole treatment to arrest cells at M phase. (B) GST pull-down assay was performed using GST-tagged wild-type (WT) or mutant (MUT) PLK1 PBD (upper panel). Equal loading of kinase substrates is indicated by Coomassie Blue staining (lower panel). (C) Bacterially expressed CRAF was subjected to in vitro kinase assays with constitutively active (TD) or kinase-defective (KM) PLK1 mutants purified from insect cells. The phosphorylation of CRAF was observed by immunoblotting with the indicated antibodies. Ponceau S staining of the blot was used to indicate equal loading of the assays. (D) RWPE-1 cells were infected with lentivirus expressing empty vector (EV), wild-type PLK1 (WT), constitutively active T210D (TD), or kinase-dead K82M (KM) mutants. Total cell lysates were subject to immunoprecipitation with CRAF antibody and then analyzed by immunoblotting. p/t: indicates densitometric intensity ratio of phosphorylated to total CRAF. (E) Immunoblotting analysis of ERK1/2 activation (phosphorylation) in RWPE-1 cells expressing EV or PLK1 WT, TD, or KM mutants. (F) C4-2B cells were infected with lentiviral shRNA constructs that target either the 3’-UTR of endogenous PLK1 (shPLK1#1) or serve as a control. Wild-type (WT) or kinase-defective (KM) PLK1 were then re-expressed in PLK1 knockdown cells. The levels of phosphorylated and total CRAF, MEK and ERK, Fral, and ZEB1/2 were determined by Western blotting analysis. (G) C4-2B cells were arrested in M phase by nocodazole (0.1 mg/mL) treatment for 16 hr, and then subject to BI 2536 treatment for 30 min. The levels of phosphorylated and total CRAF were determined by Western blotting analysis. (H) Flag-tagged CRAF WT-, CRAF-A/A-, and CRAF-D/E-overexpressing RWPE-1 cells were subjected to immunoprecipitation with anti-Flag antibody followed by immunoblotting with the indicated antibodies. (I) Control- (EV), PLK1 WT-, PLK1 TD- and PLK1 KM-overexpressing RWPE-1 cells were treated with cycloheximide (CHX: 20 μg/ml) for the indicated times, and the CRAF protein levels were monitored by immunoblotting (left panel). Quantification of the endogenous CRAF protein levels relative to β-actin expression is shown in the right panel. The data are presented as the mean ± s.e.m. (J) PLK1 WT-, and PLK1 KM-overexpressing RWPE-1 cells were treated with cycloheximide (CHX: 20 μg/ml) along with vehicle (DMSO), MG132 (2.5μM), or chloroquine (CLQ, 50 mM) for the indicated times, and level of CRAF protein was examined by immunoblotting. (K) Flag-tagged CRAF WT-, CRAF-S621A-, CRAF-S621D-, CRAF-A/A-, and CRAF-D/E-overexpressing RWPE-1 cells were treated with cycloheximide (CHX: 20 μg/ml) for the indicated time and the level of CRAF protein was determined by immunoblotting. (L) A proposed diagram of a novel signaling cascade that mediates PLK1-dependent induction of EMT and cell motility.