Figure 2(A) Endogenous SOX9 protein turnover in the presence of cycloheximide following RNAi depletion of FBW7α, β or γ. HEK293 cells were transfected with 20 nM of non-targeting scramble RNA or those specifically targeting FBW7α, β or γ for 72 hours prior to chase with addition of 100 ng /mL cycloheximide. Immunoblot of Cyclin E, an established SCF-FBW7 substrate, was used to assess the efficacy of RNAi-mediated depletion of FBW7 protein with GAPDH served as protein loading control.(B) Immunofluorescence staining of D324MED medulloblastoma cells showing nuclear co-localization of HA-SOX9 WT (AlexaFluor 568; Red) and FLAG-FBW7α (AlexaFluor 488; Green). The cell nuclei were counter-stained with DAPI (blue). Image set is representative for multiple image fields taken at 40x objective magnification. Scale bar indicate 20 µm.(C) Western blotting of HEK293 endogenous SOX9 proteins 24 hours following forced expression of 100, 250, 500, and 1000 ng of plasmid expressing FLAG-FBW7α. Changes in SOX9 protein level in the representative blots were analysed relative to GAPDH using ImageJ. The set of blots shown is representative from 3 independent experiments.(D) Expression of FLAG-FBW7α enhanced HA-SOX9-WT protein turnover, which can be attenuated with addition of 10 µM MG-132 (MG) in HEK293 cells. By contrast, forced expression of FLAG-FBW7α did not enhance HA-SOX9-T236A and -T236A/T240A protein turnover over the course of 4 hours protein chase. Protein loading for each sample was indicated by the level of β-actin protein. The set of blots shown is representative from 4 independent experiments.(E) Treatment of HEK293 cells with increasing concentration of BIO, a GSK3α/β inhibitor, increased HA-SOX9-WT protein level in dose-dependent manner. HA-SOX9-WT and FLAG-FBW7α were co-expressed in HEK293 overnight prior to treatment with various concentration of BIO for 4 hours. Whole cell lysates were collected for Western blotting with protein loading for each sample adjusted to GAPDH protein level. The set of blots shown is representative from 2 independent experiments.(F) The RNAi depletion of GSK3ß decreased FBW7α-induced HA-SOX9-WT turnover in HEK293 cells. The HA-SOX9-WT protein turnover was examined in the presence of 100 ng/mL cycloheximide, 48 hours following si-GSK3ß (20 nM) transfection, 24 hours after co-expression of HA-SOX9-WT and FLAG-FBW7α. Immunoblots of GSK3ß demonstrated depletion of GSK3ß protein with the siRNA. Changes in SOX9 protein level in the representative blots were analysed relative to GAPDH using ImageJ. The set of blots shown is representative from 3 independent experiments.(G) Forced FLAG-FBW7α-WT expression promoted poly-ubiquitylation of endogenous SOX9 in HEK293, which accumulated further in the presence of 10 µM MG132. Expression of non-functional FBW7α lacking the F-box domain (ΔF), or those containing R465A mutation did not induce SOX9 poly-ubiquitylation in vivo. Ubiquitylation assay was performed under denaturing condition to disrupt non-covalently linked ubiquitin as described in the Materials Methods. Expression of various FLAG-FBW7α constructs and endogenous SOX9 protein were examined in the whole cell extract (WCE). GAPDH protein was used to indicate total protein loading with protein loading. The set of blots shown is representative from 4 independent experiments.(H) Reconstitution of SOX9 ubiquitylation reaction by FLAG-FBW7α in vitro. Immobilized-IVT HA-SOX9 WT were incubated with complete ubiquitylation reaction mixture containing either the eluted FLAG-FBW7α-WT or the ΔF mutant for 60 minutes at 37◦C. Reaction mixture lacking the UbE1, an E1 ubiquitin-activating enzyme, or UbcH3, an E2 ubiquiting-conjugating enzyme, served as negative control for the reaction. SOX9 ubiquitylation was assessed following elution of the protein from the immobilized beads under denaturing condition as described in Materials Methods. The set of blots shown is representative from 3 independent experiments.