Western blot analysis of tyrosine phosphorylated c-Abl and Bcr-Abl was performed following immunoprecipitation of K562 cell lysates using a c-Abl antibody, from untreated cells (left lane) or cells treated with 10uM of the tyrosine kinase inhibitor Gleevec (right lane) for 24 hours. The captured immune complexes were resolved on a 4-20% Tris-HCl polyacrylamide gel, transferred to a PVDF membrane, and blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with a Phosphotyrosine monoclonal antibody (Product # MA4-003) at a dilution of 1:250 overnight rotating at 4°C, washed in TBST, and probed with a goat anti-mouse IgG-HRP secondary antibody (Product # 31430) at a dilution of 1:20,000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34075).

Immunoprecipitation of Phosphotyrosine (p-Tyr) was performed on K562 cell lysates from untreated cells (left lane) or from cells treated with 10uM of the tyrosine kinase inhibitor Gleevec (right lane) for 24 hours. Antigen-antibody complexes were formed by incubating 500ug of whole cell lysate with 2ug of a p-Tyr monoclonal antibody (Product # MA4-003) overnight on a rocking platform at 4°C. The immune complexes were captured on 50ul Protein A/G Agarose (Product # 20421), washed extensively, and eluted with Lane Marker Reducing Sample Buffer (Product # 39000). Samples were resolved on a 4-20% Tris-HCl polyacrylamide gel, transferred to a PVDF membrane, and blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with a c-Abl polyclonal antibody at a dilution of 1:1000 overnight rotating at 4°C, washed in TBST, and probed with goat anti-rabbit IgG-HRP secondary antibody (Product # 31460) at a dilution of 1:20,000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34075).