Impact of KCTD19 mutations on protein stability and protein degradation by ubiquitination in vitro. (A) Immunoblotting (IB) analysis revealing the effects of the mutations on KCTD19 protein expression levels in HEK293T cells transfected with wild type (WT) or mutant (M) plasmids. WT, M1, M2, and M3 represent plasmids encoding WT KCTD19-3×FLAG, p.E210K_KCTD19-3×FLAG, p.P298L_KCTD19-3×FLAG, and p.G770D_KCTD19-3×FLAG, respectively. The histograms below indicate the relative expression levels of KCTD19. The data are shown as means and standard errors of the mean. *P < 0.05, ***P < 0.001. (B) Detection of KCTD19 ubiquitination in HEK293T cells cotransfected with WT/mutant KCTD19-3×Flag and pCMV-Ubiquitin (Ub)-HA. Top: anti-Flag immunoprecipitation (IP) pellets immunoblotted with anti-Ub for Ub-KCTD19 conjugates (upper), or anti-KCTD19 to normalize the loading amounts (lower). Bottom: IB of KCTD19 protein levels in respective cell lysates, with GAPDH serving as a loading control.