SOCS3 is hyperphosphorylated by JAK2 V617F. (A) 293T cells were transfected with EPOR and SOCS3 alone or in combination with JAK2 WT or JAK2 V617F. Cells were treated with or without EPO (50 U/mL) for 15 minutes prior to lysis. Whole-cell lysates were immunoblotted with anti-JAK2 to detect JAK2 or anti-FLAG to detect SOCS3. The remaining lysates were immunoprecipitated with anti-SOCS3 clone 008 and resulting precipitates were immunoblotted with antiphosphotyrosine (4G10). (B) 293T cells were transfected with EPOR and JAK2, V617F, or FLAG-SOCS3 alone or combinations of JAK2 SOCS3 or V617F SOCS3 and analyzed as in panel A. (C) PBMCs were isolated from blood samples from a control patient and 2 individual patients with PV (patient nos. 1 and 2) homozygous for JAK2 V617F by Ficoll density gradient centrifugation. Cells were washed twice with PBS before being lysed in Brij buffer. Whole-cell lysates and anti-SOCS3 immunoprecipitates were immunoblotted with antiphosphotyrosine and reprobed with anti-SOCS3.