Fig. 2: Phosphorylation of Pin1 by Plk1 enhances its interaction with UPS34.

a Immunoblot analysis of the serine phosphorylation (pSer) status of Pin1 in GSCs and NSTCs. b Immunoblot analysis of Plk1 protein levels in GSCs and NSTCs. c Immunoblot analysis of Pin1 protein levels in GSCs treated with the Plk1 inhibitor SBE 13 HCl (20 μM) or DMSO for 24 h. d Immunoblot analysis of the serine phosphorylation (pSer) status of Pin1 in GSCs after inhibition of Plk1. Cells were treated with SBE 13 HCl (20 μM) or DMSO for 12 h. MG132 (10 μM) was added 6 h before harvest to obtain enough Pin1 proteins after Plk1 inhibition. e Co-immunoprecipitation to determine the interaction between Pin1 and USP34 in GSCs after inhibition of Plk1. Cells were treated with SBE 13 HCl (20 μM) or DMSO for 9 h and harvested. MG132 (10 μM) was added 6 h before harvest to obtain enough Pin1 proteins after Plk1 inhibition. f Immunoblot analysis of the poly-ubiquitination status of Pin1 in GSCs after inhibition of Plk1. Cells were treated with SBE 13 HCl (20 μM) or DMSO for 9 h and harvested. MG132 (10 μM) was added 6 h before harvest. g Co-immunoprecipitation to determine the interaction between USP34 and the wild type (WT) Pin1 or the Pin1-S65A mutant. Serine 65 (S65) at Pin1 was mutated to alanine to construct the Pin1-S65A mutant. Flag-tagged Pin1-WT or Pin1-S65A was overexpressed in 293 T cells. Expression of the endogenous Pin1 was disrupted in 293 T cells with an shRNA targeting the 3’-untranslated region (3’-UTR) of Pin1 (shPin1-3’UTR). Overexpression of ectopic Pin1 and disruption of endogenous Pin1 were achieved by PEI-mediated transfection. h Immunoblot analysis of the poly-ubiquitination status of Pin1-WT or Pin1-S65A proteins in 293 T cells. Flag-tagged Pin1-WT or Pin1-S65A was introduced into 293 T cells along with shPin1-3’UTR. Cells were treated with MG132 for 6 hours before harvest. The blotting experiments were repeated at least three times with biological replicates (a–h). Source data are provided as a Source Data file.