Tyrosinephosphorylation of BLNK by Syk is responsible for the binding of BLNK to the Btk-SH2 domain. Flag-BLNK was expressed with or without Syk in 293T cells and the cell extracts were incubated with the GST fusion protein of the Btk-SH2 domain. The binding of Flag-BLNK to GST proteins was detected by immunoblotting the cellular proteins bound to the fusion protein beads with the anti-Flag antibody (top panel). Expression of Flag-BLNK (second) or Syk (bottom) in the lysates was detected by immunoblotting with the anti-Flag antibody or the anti-Syk antibody. The tyrosinephosphorylation of Flag-BLNK by Syk was confirmed by immunoprecipitation (IP) with the anti-Flag antibody followed by immunoblotting with the antiphosphotyrosine antibody 4G10 (third).