GARP protein is increased in murine PMF and is expressed exclusively on MKs and Tregs. (A) Schematic representation of the experimental design. BM cells were isolated from 5-FU-treated donor mice, transduced with a retrovirus encoding MPL^W508A or MPL^WT and transplanted into lethally irradiated recipient mice (n = 5 mice/group). BM cells and splenocytes were isolated from MPL^W508A or MPL^WT control mice 34 days after transplantation. (B) Western blot analyses of BM cells and splenocytes with antibodies against GARP, pSTAT5, or β-actin. Bar graphs show quantification of enhanced chemiluminescence signals. P values were calculated using a two-tailed Student t test. (C) Proportions of GARP⁺ Tregs and GARP⁺ MKs in live single cells, as determined by flow cytometry. Fold differences between mean proportions in MPL^W508A and MPL^WT mice are indicated above bar graphs. (D) Histograms showing GFP expression on Tregs and MKs in the BM and spleen from 1 representative MPL^W508A mouse. Cells from a C57BL/6 mouse that did not undergo transplantation were used as negative controls. The experiment is representative of at least 3 other independent experiments.