Mpl W272R and K39N/W272R are absent from the cell surface and do not respond to ligand stimulation. (A) Western blot results for total Mpl protein and phosphorylated (p) signaling partners in cell lysates prepared from transfected Ba/F3 cells with and without Tpo stimulation (50 ng/mL, 10 minutes, 37°C). Labels at the top indicate WT or mutant Mpl^mNG constructs for each cell line tested. (B) Representative confocal images of the live Ba/F3 cells used for western blot characterization in panel A. Closed arrowhead symbols indicate the presence of surface Mpl in cells expressing WT or K39N Mpl. Open arrowhead symbols point to the absence of a clearly defined plasma membrane outline in cells expressing W272R or K39N/W272R mutant Mpl. (C) Image analysis of human UT-7 cells co-expressing Mpl^mNG WT or mutant proteins and the ER-resident protein calreticulin (CRT) fused to TagRFP-T (CRT^TagRFP-T). Co-localization of both fluorescent markers was assessed by using Pearson’s analysis of dual-channel confocal images from at least 20 cells for each condition. Means ± standard error of the mean are shown, and pairwise statistical analyses using unpaired Student t test are represented by horizontal bars. Representative images of each cell population are shown at the bottom of the panel. (D) Co-immunoprecipitation (IP) of mutant Mpl proteins with ER-resident proteins CRT and calnexin (CANX) in stably transfected UT-7 cells. Upper bands in the WT and K39N (KN) lanes represent fully glycosylated receptors, indicative of maturation in the Golgi. Scale bars = 5 µm. *P < .05; ***P < .0001. n.s., not significant.