(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. 