Fig. 6

CDK5 modulates protein stability of TPX2. a The putative consensus CDK5 phosphorylation sites of TPX2 proteins in different organisms. Alignment of TPX2 proteins sequence from different organisms indicated that the CDK5 phosphorylation site is conserved in different organisms. b Protein complex between TPX2 and CDK5. Cell lysates were immunoprecipitated (Co-IP) with GFP antibody followed by IB with TPX2 antibodies. c In vitro phosphorylation assay of recombinant wild-type TPX2 and non-phosphorylatable TPX2 (S486A) with activated CDK5/P25. Immunoblot detecting the phosphorylation of purified GST recombinant TPX2, and non-phosphorylatable TPX2-S486A mutation using an antibody against thiophosphate esters (Top). Coomassie blue stain (bottom) and the immunoblot with anti-GST antibodies (middle) reveals that both wild-type TPX2 and non-phosphorylatable TPX2(S486A) were loaded in similar amounts. The asterisk indicated that the f​ull length of TPX2-WT and TPX2-S486A. d Immunoblotting of TPX2 are shown in Huh7 cells transfected with GFP-CDK5-CMV, GFP-CDK5, GFP-CDK5-KD constructs, GAPDH serve as control. The average of TPX2 protein levels with Mean values±SEM are presented in the right panel, One Way ANOVA on Ranks,*P < 0.05; **P < 0.01;***P < 0.001. e Similar to d, TPX2 protein levels in shCDK5 SMMC-7721 cells were shown, t test,***P < 0.001. f Treatment of SMMC-7721 cells with 40 μM roscovitine inhibited TPX2; The average of TPX2 protein levels with Mean values±SEM are presented in the right panel,t test,**P < 0.01. g Relative mRNA levels of TPX2 were measured in CDK5 overexpressed Huh7 cells by Real-Time qPCR, t test,***P < 0.001. h Relative mRNA levels of TPX2 were measured in shCDK5 SMMC-7721 cells by Real-Time qPCR, t test,***P < 0.001. i Degradation of TPX2 protein in shcon and shCDK5 SMMC-7721 cells after treatment by CHX in in time gradient, relative TPX2 protein level over the time was shown in the right panel. j Degradation of TPX2 protein in CDK5 overexpression Huh7 cells after treatment by CHX in in time gradient, relative TPX2 protein level over the time was shown in the right panel. k Degradation of TPX2 protein in GFP-CDK5-WT(wildtype), mutant GFP-TPX2 (S486A), phosphor mimic mutant GFP-TPX2 (S486D) after treatment by CHX in in time gradient. l In vitro ubiquitination assay was performed in 293 T cells transfected with TPX2-WT,TPX2-S486A, TPX2-S486D, Immunoblotting with anti-HA antibody(above) and anti-TPX2 antibody (below) were shown. m Treatment of SMMC-7721 cells with 20 μM tamoxifen inhibited TPX2; The average of TPX2 protein levels with Mean values±SEM are presented in the right panel, t test,***P < 0.001