Downmodulation of MPL in platelets of patients with MPN. (A) Gating strategy used for analysis of fixed and permeabilized platelets by flow cytometry. (B,D) Representative histograms showing surface (B) or total (D) MPL staining with an anti-MPL antibody in platelets from patients with MPN (red histograms) or same-day healthy control (HC) platelets (blue histograms) in unfixed (B) or fixed and permeabilized (D) platelets. (C,E) MFI values of the surface (C; n = 12 for Del52 and n = 7 for Ins5 samples) and total (E, n = 19 for Del52 and n = 7 for Ins5 samples) MPL staining on platelets. Each dot in (C) and (E) represents the MFI value for an individual MPN or HC platelet sample, whereas the lines connect pairs of samples of HC and patients with MPN that were processed in parallel. Statistical significance indicated by P values was determined using GraphPad Prism and paired t test analyses. (F) Representative blots showing MPL and mutant CRT protein levels in the lysates of platelets from HC or patients with MPN subjected to SDS-PAGE under reducing conditions, followed by immunoblotting. Anti-MPL and anti-CRT(C_mut) antibodies were used for the detection of MPL and mutant CRT proteins, respectively. GAPDH blot shows the relative loading of the lysates. The heterogeneity of CD41 expression in the representative panel shown in A most likely relates to the limiting amount of antibody, as similar results were obtained based on CD41^hi-gating vs gating on all single cells. FSC-A, Forward Scatter-Area; FSC-H, Forward Scatter-Height; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SSC-A, Side Scatter-Area; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.