JAK2 gene amplification, JAK2 mRNA expression, and Western blot analysis. (A) JAK2 gene amplification in MedB-1 cells. DNA prepared from MedB-1 cells, L428 cells, and autologous fibroblasts from the parental tumor of MedB-1 was analyzed by real-time PCR with PCR primers specific for an DNA fragment within exon 5 of the JAK2 gene and normalized on β2-microglobulin gene exon 2. JAK2 DNA contents in MedB-1 and L428 are related to those in fibroblasts calculated as 100%. (B) JAK2 mRNA expression in MedB-1 cells, L428 Hodgkin cells, and Ramos cells. Expression is related to that in peripheral B-lymphocytes. Error bars indicate the standard deviation of 3 independent experiments. (C) Western blot analysis of JAK2/STAT5 and its phosphorylated forms. JAK2 and STAT5 protein expression and its phophorylation were measured in total protein extracts of MedB-1, L428, and Ramos cells by immunoblotting; 20 μg total protein was subjected per lane. JAK2/STAT5 and phospho-JAK2 (*)/phospho-STAT5 (←), respectively, were detected simultaneously with the appropriate antibodies; β-actin was used as loading control.
