The c-MPL F104S, but not the P106L-mutated receptors heterodimerize with the WT receptor. (A) Representative confocal images of transiently cotransfected HeLa cells. The (Aa, Ad, and Aj) c-Mpl WT-GFP or (Ag) c-Mpl R102P-GFP, respectively, harboring a GFP expression sequence in frame at the C-terminus and (Ab) c-Mpl R102P-FLAG, (Ae and Ah) F104S-FLAG, or (Ak) P106L-FLAG mutated constructs with a FLAG-tag sequence in frame at the C-terminus were cotransfected with equal amounts of DNA and fixed 48 hours later. The FLAG-tag was visualized with a mouse anti-FLAG M2 primary antibody and an anti-mouse secondary antibody linked to Alexa 568 as described in “Materials and methods.” (Aa, Ab, and Ac) Cotransfection of c-Mpl WT-GFP and c-Mpl R102P-FLAG. (Ad, Ae, and Af) Cotransfection of c-Mpl WT-GFP and c-Mpl F104S-FLAG. (Ag, Ah, and Ai) Cotransfection of c-Mpl R102P-GFP and c-Mpl F104S-FLAG. (Aj, Ak, and Al) Cotransfection of c-Mpl WT-GFP and c-Mpl P106L-FLAG. Panels Ac, Af, Ai, and Al represent the respective overlays of the cotransfection experiments. Images were acquired using a spinning-disc confocal microscope with a ×100 oil immersion objective. Bars represent 10 µm. Cells depicted represent the subcellular distribution pattern seen in >90% of the transfected cells. (B) Pearson’s correlation coefficient of the cotransfection experiments described in (A) (n = 5). The coefficient was acquired with the Volocity 5.5 software using the automatic threshold function. The ROI was defined as a 10 × 10-µm square with the Golgi set as the middle for the ROI. *P = .013; ****P < .0001.