Retroviral gene signaling activity, mRNA expression level, and genomic integration from JAK2^V617F virus-recipient mice. (A) Western blot analysis from spleen cells demonstrates constitutive STAT5a and ERK1/2 pathway activation in JAK2^V617F recipients in contrast to JAK2^WT recipient mice. Total and phosphorylated (p) Stat5a and ERK1/2 are shown in the absence of added growth factor (NS) or the presence of Epo and IL-3. (B) Ratio between total (retroviral and endogenous) and endogenous JAK2 mRNA. Total JAK2 mRNAs were quantified by QTPCR against tubulin (Table 1). Endogenous level was determined from empty virus-recipient mice. Results are mean values from 3 to 4 mice, studied between 3 and 6 months after transplantation with triplicated RNA measurements. WT and E are pooled levels from JAK2^WT or empty virus recipient. MP1 and MP2 or MS1 and MS2 are pooled from primary or secondary JAK2^V617F-recipient mice, respectively. (C) Identification of proviral insertions in the genome of spleen (SP) and BM samples from JAK2^WT (WT) or JAK2^V617F virus primary-(MP-A to MP-D) or secondary (MS-A)-recipient mice. Animals were studied at the indicated day after transplantation (in bracket). Proviral integrants were detected using Southern blot analysis with a GFP probe from EcoRI- or BamH1-digested DNA exhibiting 1 enzymatic cut in the proviral DNA and in the genomic DNA. Bands indicate individual proviral integrants. MP-A, MP-B, and MP-D developed a high-grade fibrosis, and MP-E developed a low-grade fibrosis. No fibrosis was detected for MP-C or WT.