Cell surface localization of c-Mpl WT and mutants. (A-B) Representative confocal images of HeLa cells transiently transfected with (Aa-Ac, Bd-Bf) the HA-c-Mpl WT-GFP, (Ag-Ai, Bj-Bl) the HA-c-Mpl R102P-GFP, (Am-Ao, Bp-Br) the HA-c-Mpl F104S-GFP, and (As-Au, Bv-Bx) the HA-c-Mpl P106L-GFP receptor constructs, respectively. (A) An extracellular HA-tag introduced behind the secretory leader of the c-Mpl receptor 3′ of codon 26 was used to detect plasma membrane expression without Triton X-100 permeabilization (Ab, Ah, An, and At). (B) Cells permeabilized with 0.1% Triton X-100 served as a control for the accessibility of the HA-tag and the identical distribution of the constructs harboring the HA-tag (Be, Bk, Bq, and Bw). The HA-tag was visualized with a mouse anti-HA primary antibody and an anti-mouse secondary antibody linked to Alexa 568 as described in “Materials and methods.” Images were acquired using a Perkin Elmer spinning-disc confocal microscope with a ×100 Nikon oil immersion objective. Bars in the overlay pictures represent 10 µm. Cells depicted represent the subcellular distribution pattern seen in >90% of the transfected cells. (C-D) Fluorescent cell sorting of transiently transfected HeLa cells is shown. The c-Mpl WT, R102P, F104S, and P106L constructs harbored the HA-tag at the N-terminus behind the secretory leader. The mCherry plasmid was cotransfected as a control for transfection efficiency. After the harvest, cells were incubated with anti-HA primary antibody followed by secondary anti-mouse IgG conjugated to AlexaFluor 488. Cells transfected with c-Mpl WT served as negative controls. Cells were gated for positive mCherry signal and analyzed as described in “Materials and methods.” (D) Results and standard deviations represent the median of 3 independent experiments. For quantification, HA-c-Mpl WT surface expression was set as 1.0. n.s., nonsignificant.