Phosphorylation of Lu/BCAM in RBCs and K562 cells. Normal or PV RBCs were incubated with 32P orthophosphate in the absence (A) or presence (B) of forskolin, and Lu/BCAM was immunoprecipitated using mAb against Lu/BCAM. After elution, SDS-PAGE, and transfer to nitrocellulose membrane, the phosphorylated proteins were detected using a PhosphorImager. As previously reported, no phosphorylation was detected in the normal RBCs, while in the 6 patients with PV, the Lu/BCAM long isoform was phosphorylated in the absence of any stimulation. Western blot (WB) using anti-Lu rabbit antibody 602 showed that equivalent amounts of Lu protein were immunoprecipitated for each patient in the absence or presence of forskolin in panel B. (C) Phosphorylation of recombinant Lu/BCAM gp in K562 cells. Recombinant JAK2 WT or 617V>F were transiently expressed in K562-Lu/BCAM cells and detected by Western blot (WB; top row). Expression of recombinant Lu/BCAM was tested in cell lysates by Western blot (second row). Phosphorylation of Lu/BCAMgp was analyzed after immunoprecipitation (IP; third panel), and total immunoprecipitated Lu/BCAM proteins were revealed by Western blot (bottom row). Results are representative of 3 independent experiments. The phosphorylation in K562 cells tranfected with 617V>F was 3.7-fold (± 1.1-fold) higher, while Lu/BCAM protein was doubled (2.2-fold ± 0.3-fold) compared with K562 cells transfected with WT JAK2.
