Fig. 4: GSK-3β inactivation-mediated dephosphorylation of cyclin D1 facilitates FBXO32-induced cyclin D1 protein ubiquitination and stabilization.
From: F-box protein FBXO32 ubiquitinates and stabilizes D-type cyclins to drive cancer progression
a 293T cells were co-transfected with Flag-FBXO32 and WT HA-cyclin D1, or various HA-cyclin D1 mutants (T286A, T288A) or a Vector control for 48 h, followed by co-IP assay. b 293T cells were co-transfected with Flag-FBXO32 and His-Ub together with WT HA-cyclin D1, various HA-cyclin D1 mutants (T286A, T288A) or a Vector control for 48 h, then the cells were lysed to detect ubiquitinated cyclin D1. c, d Huh7 and CFPAC-1 cells were co-transfected with Flag-FBXO32 and WT HA-cyclin D1 or various HA-cyclin D1 mutants for 48 h, followed by treatment with 100 μg/mL CHX for the indicated times, then cyclin D1 was determined by western blotting and quantified by Image J software. e, f After co-transfection with His-Ub and HA-cyclin D1T286A (or HA-cyclin D1T288A) together with Flag-FBXO32 or a Vector control for 48 h, 293T cells were lysed for ubiquitination assay. g, h Huh7 cells were co-transfected with HA-cyclin D1T286A (g) or HA-cyclin D1T288A (h) and Flag-FBXO32 or a Vector control for 48 h, followed by a CHX pulse-chase assay. i 293T cells were co-transfected with HA-cyclin D1 and His-Ub together with Flag-FBXO32 or a Vector control for 36 h, followed by treatment with GSK-3β inhibitor TWS119 (5 μM), DYRK1B inhibitor AZ191 (1 μM) or MEK1/2 inhibitor trametinib (100 nM) for another 12 h, followed by ubiquitination assay. j After co-transfected with HA-cyclin D1, His-Ub and Flag-FBXO32 together with GSK-3β or negative control siRNA for 48 h, 293T cells were lysed for ubiquitination assay. k 293T cells were co-transfected with HA-cyclin D1, His-Ub and Flag-FBXO32 together with WT GSK-3β or various GSK-3β mutants, followed by ubiquitination assay. l 293T cells were transfected with WT GSK-3β, or various GSK-3β mutants, or negative control vector for 48 h, followed by GSK-3β activity measurement. (n = 3 independent experiments). Representative immunoblot was shown from 3 biologically independent experiments (a–k). Data are presented as mean ± SEM, and P-values were calculated using one-way ANOVA with Dunnett’s (c, d) or Tukey’s (l) multiple comparisons or two-tailed unpaired Student’s t-test (g, h). Source data are provided as a Source Data file.
