MPLS204P does not exhibit cellular trafficking defects. (A) MPL cell-surface expression analysis by flow cytometry. UT7 cells transduced with either the HA-tagged MPLWT or HA-tagged MPLS204P receptor were incubated with an anti-HA antibody coupled with PE. Histograms show equivalent GFP expression (left) monitoring the total MPL levels and HA-PE labeling (right) corresponding to similar cell-surface expression of MPLWT and MPLS204P. (B) Analysis of the different forms of MPL. UT7 MPLWT and MPLS204P cell extracts were treated with EndoH (500 U, overnight at 37°C) and western blot analysis was performed using anti-HA antibody. The different forms of MPL with the EndoH-resistant mature form corresponding to a 85-kDa band which is equivalent in both cell lines are shown. Samples were run on the same gel and vertical lines have been inserted to indicate a repositioned gel lane. (C) Localization of MPL in the ER and (D) in the Golgi apparatus. MPLWT and MPLS204P UT7 cells were analyzed by immunofluorescence using anti-CALR (ER marker), anti-GM130 (Golgi apparatus marker), and anti-HA (MPL detection) antibodies. (E) Internalization of the MPLWT and MPLS204P after TPO binding. These experiments were performed with Ba/F3 cells expressing either MPLWT or MPLS204P. MPL cell-surface expression was studied by flow cytometry after TPO addition. Results are means ± SD of 3 independent experiments. SD, standard deviation.