Fig. 1: FBXO32 directly interacts with cyclin D1.
From: F-box protein FBXO32 ubiquitinates and stabilizes D-type cyclins to drive cancer progression
a 293T cells were co-transfected with His-Ub and wild-type (WT) HA-cyclin D1, or various HA-cyclin D1 mutants (T286A and T288A), or a Vector control for 48 h. Then the cells were lysed with ubiquitination assay lysis buffer, followed by pull-down using Ni-NTA beads, and the precipitates were analyzed by western blot. b Schematic of the screening workflow to identify FBXO32 as a protein candidate interacting with dephosphorylated cyclin D1. c Co-immunoprecipitation (Co-IP) assay was utilized to determine the interaction between endogenous FBXO32 and cyclin D1 in Huh7 cells. IgG indicates the control antibody group. d Co-IP analysis of the interaction between Flag-tagged FBXO32 and HA-tagged cyclin D1 in Huh7 cells transfected with the indicated expression constructs. e Huh7 or CFPAC-1 cells were transfected with Flag-FBXO32 plasmid or control Vector for 48 h. Immunofluorescence assay was utilized to measure the colocalization of FBXO32 and cyclin D1. Hoechst was used to stain DNA. Scale bar, 10 μm. f Huh7 cells transfected with HA-cyclin D1 construct were lysed and incubated with GST or GST-FBXO32 conjugated to beads. Pull-down samples and 5% of the input were analyzed by western blot and Ponceau S staining. g SPR assay revealed the kinetic interaction between His-tagged recombinant human FBXO32 and cyclin D1. h 293T cells were co-transfected with WT HA-cyclin D1 and various domain-deleted Flag-FBXO32 plasmids for 48 h. MG132 (10 μM) was added to incubate with the cells for 4 h before being harvested. Then co-IP assay was performed using an anti-HA antibody to pull down HA-tagged cyclin D1, followed by western blot analysis. i WT HA-cyclin D1 and cyclin D1 truncation mutants were co-transfected with Flag-FBXO32 into 293T cells for 48 h. MG132 was added to incubate with the cells for 4 h before being harvested. Then, cells were collected for the co-IP assay using anti-Flag antibody, followed by immunoblotting. Representative immunoblot was shown from 3 biologically independent experiments (a, c, d, f, h, i). Source data are provided as a Source Data file. WCE whole cell extraction, OE overexpression.
