Lnk interacts differently with JAK2-WT and V617F. (A) Schematic representation of GST-Lnk fusion proteins used; R364M mutation is indicated with an asterisk (*). (B) UT7/Mpl stimulated as previously indicated were incubated with GST alone or different GST-fused Lnk domains. (C) COS7 cells transiently transfected with either WT (left panel) or JAK2-V617F (right panel) forms by the polyethyleneimine (PEI; Sigma-Aldrich) method were harvested 48 hours after transfection and lysed as described in methods section. Anti-JAK2 Western blot confirms similar expression of both JAK2 forms. Cell lysates were incubated with GST alone or different GST-fused Lnk domains. Immunoblotting with anti-p-Tyr, anti-phospho-JAK2 and anti-JAK2 antibodies allowed detection of bound activated JAK2 protein. Anti-JAK2 immunoprecipitation and total cell lysate (TCL) are shown as control for JAK2 size. Anti-GST blots are shown as control of equal GST proteins used in the assay, asterisk (*) in panels B-C marks NPH GST protein. All experiments have been performed at least 3 independent times. White space gaps in panels B and C indicate repositioned gel lanes.