Different subcellular distribution and Golgi localization of c-Mpl WT and mutants. (A) Representative confocal images of transiently transfected HeLa cells. The c-Mpl-GFP minigenes harboring the green fluorescent protein (GFP) expression sequence in frame at the C-terminus were transfected as follows: (Aa-Ac) c-Mpl-WT-GFP, (Ad-Af) c-Mpl-R102P-GFP, (Ag-Ai) c-Mpl-F104S-GFP, and (Aj-Al) c-Mpl-P106L-GFP. The Golgi complex was visualized by a primary mouse antibody against of the Golgi-resident protein GM-130 and a secondary anti-mouse antibody coupled to Alexa 568 (Figure 4A, middle panels Ab, Ae, Ah, and Ak). The panels on the right (Ac, Af, Ai, and Al) represent overlays with the c-Mpl mutants and the Golgi marker. Interfering influence of the GFP-fusion protein on the subcellular distribution was excluded by FLAG-tagged (Am) c-Mpl WT-FLAG and (An) c-Mpl P106L-FLAG constructs harboring the FLAG-Tag at the C-terminus. The FLAG-tag was visualized by a mouse anti-FLAG M2 antibody and a secondary anti-mouse antibody coupled to Alexa 568 as described in “Materials and methods.” The nucleus was visualized by 4,6 diamidino-2-phenylindole staining. Bars in the overlay pictures represent 10 µm. Images were acquired using a Perkin Elmer spinning-disc confocal microscope with a ×100 Nikon oil immersion objective. Cells depicted are representative of the subcellular distribution pattern seen in >90% of the transfected cells. (B). Pearson’s correlation coefficient for the cotransfection experiments described in (Aa-Al) (n = 5). The coefficient was acquired from the Volocity 5.5 software using the automatic threshold function. The complete depicted panel was defined as region of interest (ROI) for the measurements.