Expression of FKBP51 in normal and IMF MKs.
(A) Immunofluorescence localization of FKBP51 in normal MKs. Normal MKs were cultured from CD34+ cells in the presence of TPO and SCF. After cytocentrifugation, cells were fixed, permeabilized, and labeled with an anti-FKBP51 mAb. TRITC-labeled donkey antimouse F(ab′)2 was used as secondary antibody. Cells were counterstained with DAPI (4,6 diamidino-2-phenylindole). Localization of FKBP51 in the different MK samples (in red) was analyzed using a fluorescence microscope. A cytoplasmic labeling was observed with a peculiar pattern, which could correspond to the Golgi apparatus. Original magnification, × 1000. (B) FKBP51 expression in normal and pathologic MKs studied by Western blot. Protein extracts from spontaneously growing MKs from 2 patients (P5 and P4) and one normal leukapheresis product, CD34+cell-derived MKs (cultured in presence of TPO), were separated by SDS-PAGE. Immunoblotting was performed with the anti-FKBP51 antibody, anti–GpIIb-IIIa antibody, and an antiactin antibody in the same membrane. Ratios of FKBP51/GpIIb and FKBP51/actin are illustrated as well as GpIIb/actin ratio. An increase in the FKBP51/GpIIb and FKBP51/actin ratios was observed in the 2 samples derived from PMF, whereas the GpIIb/actin was almost constant. (C) Differential FKBP51 protein expression in normal and IMF MKs by flow cytometry. Spontaneously growing MKs from IMF patients and normal MKs were doubly labeled with an anti-VWF polyclonal antibody and an anti-FKBP51 mAb by indirect immunofluorescence. Analysis of FKBP51 expression was performed in the VWF+e gate. In panels Ci and Cii, the histogram filled in gray illustrates the control isotype(IgG-FITC). The histogram filled in black illustrates the graph of a healthy control. The unfilled curve in black line shows the graph of 2 different patients. In panel Ci, 2 other controls are shown (gray lines). The 3 control sample curves are superimposed. The 2 illustrated patient MKs express a low but significant increase in FKBP51 expression.
