Constitutive nuclear localization of GR in EBs generated ex vivo from PV MNCs is associated with expression of the dominant negative GRβ isoform. (A) Immunostaining for GR of EBs obtained from 1 representative ND and PV patient with and without dexamethasone (DXM). Arrowheads indicate representative nuclear localization of GR. In EBs from ND, nuclear staining for GR was observed after DXM stimulation and had the punctuated appearance expected for GRα. By contrast, in EBs from PV, GR staining of the nucleus had a diffuse DXM-independent pattern characteristic of GRβ.⁴⁰  Magnification ×40. (B) RT-PCR analyses for expression of GRα and GRβ of EBs expanded ex vivo from 5 representative ND and PV patients. The homozygous (+/+) or heterozygous (+/−) allele status of the JAK2V617F mutation of the PV patients is indicated on the bottom. Similar results were obtained with additional NDs (n = 5) and PV patients (n = 11; not shown). In all cases, the identity of the band was confirmed by sequencing (on the right). GAPDH was amplified as control. (C) WB analyses with GRα- and GRβ-specific antibodies of EBs generated from 8 additional NDs (not analyzed at the mRNA level) and from 3 PV patients (the first patient was analyzed at the mRNA level in the second lane in panel B). β-actin was analyzed as a loading control. The proteins recognized by the GRα- and GRβ-specific antibodies migrated with an apparent molecular weight of 94 and 90 kDa, respectively. (D) WB analyses for GRβ of MNCs from NDs and from PV, ET, and PMF patients (4 each). Data are representative of those observed in a total of 10 subjects per group. In panels C and D, ND and MPN patients are identified by the same unique alphanumeric codes used in Figure 6. SNP-negative and SNP-positive subjects are indicated in black and red fonts, respectively. The SNP status of PV332 (in bold) is not known.