Fig. 6
CFP1 proteins are degraded during G2-M transition in oocytes. a CFP1 degradation during oocyte maturation. GV oocytes were microinjected with mRNA encoding CFP1-GFP-HA and cultured in medium containing milrinone. Some oocytes were removed from milrinone-containing medium 3 h later and further cultured for another 3 h in milrinone-free medium. Total proteins from 100 oocytes were loaded in each lane. b MG132 blocked CFP1 degradation in maturing oocytes. GV oocytes were microinjected with mRNA encoding CFP1-GFP-HA and cultured in medium containing milrinone for 3 h and then were transferred to milrinone-free medium with or without MG132 for further culture (6 h). c Functional domains and potential CDK1 phosphorylation sites (S/T-P) of mouse CFP1. d The C-terminus-deleted CFP1 (CFP1ΔC) was not degraded in maturing oocytes. GV oocytes were microinjected with mRNA encoding CFP1-HA or CFP1ΔC-HA and cultured as in (b). e Ubiquitination levels of CFP1-HA or CFP1ΔC-HA in HeLa cells. Cells were co-transfected with plasmids expressing the indicated proteins. Ubiquitin (Ub)-bound proteins were pulled-down from cell lysates at 48 h after transfection with Flag-affinity agarose beads. f Cycloheximide (CHX) chasing experiment showing CFP1 stability. HeLa cells were transfected with plasmids expressing CFP1-HA or CFP1ΔC-HA for 48 h and then incubated with medium containing CHX (10 μM). Cells were lysed at 0, 4, 8, and 12 h after CHX treatment and subjected to western blot. g Band shift showing CFP1 phosphorylation. GV oocytes were microinjected and cultured as in (b). Some samples were pre-incubated with protein phosphatase for 2 h before western blot. h Co-IP results showing the interaction between CFP1 and CDK1. HeLa cells were co-transfected with plasmids expressing CFP1ΔC-HA and CDK1 for 48 h before immunoprecipitation. i Constitutively active CDK1 triggers CFP1 phosphorylation and degradation. GV oocytes were microinjected with mRNAs encoding CFP1ΔC-HA and Flag-CDK114A;14F and cultured in medium containing milrinone for 8 h. j Mutation of S138 and S143 abolished the CFP1 band shift in maturing oocytes. GV oocytes were microinjected with mRNAs encoding indicated CFP1 forms and cultured as in (b)
