Fig. 6.
CHIP ubiquitylates and degrades NEK10. a Cells were co-transfected with NEK10-flag, HSP70-V5 and CHIP-myc. To prevent NEK10 degradation, cells were treated with MG132 (20 µM) for 8 h before collecting. Lysates were immunoprecipitated with anti-flag or with control IgG. The precipitates and lysates were immunoblotted with the indicated antibodies. b Cells were co-transfected with NEK10-flag vectors (either wild type or T812A mutant), HSP70-V5 and CHIP-myc, serum deprived for 24 h and left untreated or stimulated with FSK. Lysates were immunoprecipitated with anti-flag. The precipitates and lysates were immunoblotted with the indicated antibodies. c Cumulative data of the experiment shown in b. The results represent the mean values ± S.E. of three independent experiments. d Lysates from cells co-transfected with NEK10-flag and CHIP (either wild type or K30A mutant) were immunoblotted with the indicated antibodies. e Cells were co-transfected with HA-ubiquitin, NEK10-flag and CHIPK30A-myc or with control plasmid (CMV). 24 h after transfection, to avoid degradation of the ubiquitinated target protein, the cells were treated with MG132 (20 µM) for 8 h and then stimulated with FSK. Lysates were subjected to immunoprecipitation with anti-flag and immunoblotted with anti-HA, anti-flag and anti-myc antibodies. f Cells co-transfected with HA-ubiquitin, NEK10-flag and siRNAs (either control siRNA or siCHIP) were serum-deprived overnight and stimulated with isoproterenol. Lysates were subjected to immuno-precipitations with anti-flag antibody. Ubiquitinated NEK10 was revealed by immunoblot with anti-HA antibodies. g Cells were transiently transfected with control or with siRNAs targeting CHIP, serum-deprived for 36 h and then left untreated or stimulated with FSK. Primary cilia were visualized by immunostaining for acetylated tubulin. Cumulative data from five independent experiments are shown. For each group a minimum of 100 cells/experiment was averaged. *p < 0.05 versus siCNT and siCHIP + FSK