Lnk interacts with JAK2-WT and V617F via its SH2 domain. (A) Platelets from healthy donor, RT and MPN patients were stimulated as in Figure 3 and immunoprecipitated with anti-Lnk antibodies. Western blot analysis with anti-JAK2 antibodies revealed associated JAK2 (top panel). Phosphorylated Lnk was detected with anti-p-Tyr antibodies (middle panel) and Lnk protein expression is shown with anti-Lnk antibodies (bottom panel). (B) Total lysates from UT7/Mpl (left panel) or HEL (right panel) cells expressing control vector or Lnk forms were stimulated with TPO (100 ng/mL) or EPO (50 U/mL), respectively, for 10 minutes and then immunoprecipitated with anti-Lnk antibodies. Western blot analysis was performed with anti-phosphotyrosine and anti-phospho-JAK2 antibodies (top right panel). Anti-JAK2 immunoprecipitations are shown as control for JAK2 size. JAK2 and Lnk protein expression were detected with anti-JAK2 and anti-Lnk, respectively. A longer exposure of the Lnk blot (left panels) shows Lnk as a doublet (arrows). IgG (Heavy chain, Hc) blot is shown as immunoprecipitation control in left panel. All shown experiments have been repeated at least 3 independent times.