c-Mpl is ubiquitinated at K₅₅₃ and K₅₇₃. (A) Schematic representation of the human c-Mpl intracellular domain, showing the position of the 2 intracellular lysine (K) residues in relation to tyrosine (Y) and box 1 and box 2. (B) Western blot analysis of total c-Mpl expression in 2 WT c-Mpl clones and 2 c-MplK_553+573R clones. (C) Cells expressing c-Mpl with a single K to R mutation (c-Mpl K₅₅₃R and c-Mpl K₅₇₃R) display normal levels of c-Mpl ubiquitination in response to Tpo stimulation. (D) c-Mpl ubiquitination after 60-minute Tpo stimulation in BaF cells expressing WT c-Mpl or c-MplK_553+573R. The data shown are representative of 3 independent experiments. (E) Biotinylation of mature, membrane-localized c-Mpl in BaF cells expressing WT c-Mpl and c-MplK_553+573R to determine normal turnover over a time period of 6 hours. The Western blot shown is representative of 3 independent experiments. The graph displays a quantitation of the turnover of mature, 85-kDa c-Mpl in the 2 cell lines as determined by densitometry, compared with time 0 (100%). Protein loading is standardized by comparison to surface Na⁺/K⁺ ATPase α1 expression. The data points represent the mean ± SE of 3 independent experiments (*P < .05, ***P < .001; Student t test).