SOCS3 enhances JAK2 V617F phosphorylation and fails to be degraded. (A) 293T cells were transfected with combinations of EPOR, JAK2, or JAK2 V617F, and FLAG-tagged SOCS1, SOCS2, or SOCS3 as indicated. Cells were treated for 30 minutes with EPO (50 U/mL) (+EPO, top 3 panels) or left untreated (−EPO, bottom 3 panels). Lysates were immunoblotted with phosphotyrosine antibody then stripped and reprobed with JAK2 antibody (top panels) or anti-FLAG to detect SOCS proteins. (B) Cell lysates from panel A were simultaneously analyzed for SOCS protein and mRNA levels. Whole-cell lysates were immunoblotted with FLAG antibody to detect SOCS proteins, while RT-PCR was performed on the total RNA using primer pairs specific for the SOCS genes, and products were separated by 2% agarose gel electrophoresis and visualized by ethidium bromide (bottom panels).