Fig. 2. IL-3 stimulates phosphorylation of paxillin tyrosine residues 31 and 118. (A) Baf3 cells were stably transfected with EGFP (vector) or EGFP-conjugated wild-type -paxillin or the -paxillin YF tyrosine phosphorylation mutants 31, 118, 31/118, 40/181, 31/40/118/181, 31/40/181/434/488 and 40/118/181/434/488. After six hours of IL-3 withdrawal, 10 ng/ml IL-3 was resupplied for 10 minutes. Immunoprecipitated paxillin (IP-Pax) was analyzed on western blots with a mixture of anti-phosphotyrosine antibodies 4G10 and PY20 (IB-PY) or anti-GFP antibody (IB-GFP). (B) Immunoprecipitated paxillin (IP:Paxillin) was treated in vitro with non-specific potato acid phosphatase (PAP) or Protein Tyrosine Phosphatase (PTP), which specifically dephosphorylates tyrosine residues and was then analyzed on western blots using anti-paxillin antibody (IB:Paxillin).