Figure 5.
PSGL-1 signals through Fgr, DAP12, FcRγ, and Syk. Bone marrow–derived neutrophils were plated on uncoated (unstimulated) or E-selectin–coated wells for the indicated time, and then lysates were prepared. (A) Lysates were immunoprecipitated with anti-Fgr, followed by immunoblotting (IB) with a phosphospecific Src antibody that recognizes the tyrosine in the catalytic domain of Fgr (n = 3). (B and C) Lysates were incubated with GST–Syk–(SH2)2 fusion protein (ppt), and bound proteins were immunoblotted with a phosphospecific DAP12 antibody (n = 3) or (D and E) immunoprecipitated with anti-Syk followed (n = 3) by immunoblotting (IB) for phosphotyrosine (PY) or Syk or ERK as a loading control.
