Retrovirally transduced MPL P106L is localized at the cell-surface membrane, but at much lower levels than MPL WT. (A) Receptor expression in transfected UT-7 cell lines: cells cultured with GM-CSF were incubated with an anti-HA antibody coupled with phycoerythrin (PE) and analyzed by flow cytometry. For similar GFP expression, MPL P106L is minimally expressed at the cell-surface membrane, unlike the clones, which present more surface receptors, although considerably less than the cell line expressing MPL WT. As already known, MPL R102P is not detectable at the cell-surface membrane. (B) Clones selection: UT-7 MPL P106L cells were grown under THPO 10 ng/mL in methylcellulose assay. Colonies responding to THPO were selected and plated in supplemented minimum essential medium alpha supplemented with GM-CSF. (C) Analysis of the mature and immature forms of MPL: UT-7 cells cultured with GM-CSF were incubated with endoglycosidase H before western blot with an anti-HA antibody. The mature form of MPL (85 kDa), which is resistant to endoglycosidase H digestion and capable of reaching the plasma membrane, is not present in UT-7 cells transduced with MPL P106L. (D) Receptor expression on Ba/F3 cells cotransfected with JAK2: Ba/F3 cells cultured with supernatant from WEHI-3B cells were cotransduced with MPL and JAK2 and analyzed by flow cytometry after anti-HA labeling. There was no increase in MPL P106L cell-surface expression, in contrast to MPL WT. At least 3 × 10⁴ cells were analyzed in all flow cytograms.