Immunoprecipitation and Western Blotting Analysis

For immunoprecipitation, the cell lysates were incubated on ice with anti-Lyn, anti-PDGFRA anti-JAK2 or anti-IL-5RA antibody (1:100-1:1000 dilutions; Santa Cruz Biotechnology, USA) for 2 h. The immune complexes were collected following incubating with protein A-agarose (Roche, USA) at 4°C for 1 h. The beads were then washed three times with washing buffer and boiled for 5 min in SDS-PAGE sample buffer. The solubilized proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane (Amersham Biosciences, Sweden), and detected by immunoblotting against antiphosphotyrosine monoclonal antibody, 4G10 (αPY) or Western blotting was performed as described previously [15 (link)]. Blots were probed with the primary antibodies against phospho-Lyn (Y396) (p-Lyn), Lyn, phospho-p85a (PI3K)/(Tyr467)(p-p85a), p85a, phospho-Akt1 (Thr308/Ser473)(p-Akt1) and Akt1 (Santa Cruz Biotechnology, USA), phospho-Stat5(Tyr 694), and Stat5 (Invitrogen, USA), phospho-MAPK p44/42(Thr 202/Tyr 204), MAPK, phospho-JAK2 (Tyr1007/1008)(p-JAK2), JAK2 and β-actin (Cell Signaling Technology, USA) followed by incubation with the secondary antibodies were used peroxidase-conjugated goat antimouse IgG or goat anti-rabbit IgG (Jackson ImmunoResearch Inc., USA) and enhanced chemiluminescent substrate. Densitometry analysis was performed on exposed films using Quantity One v4.62 software (Bio-Rad, USA).