Expression, phosphorylation status, and enzymatic activity of bcr/abl in LAMA84 and LAMA84R cells.

(A) The expression of endogenous bcr/abl and actin in extracts of LAMA84 and LAMA84R cells treated with 0.6 μM of STI571 or left untreated (control) was determined by Western blot analysis using the monoclonal anti-abl and the polyclonal anti-actin antibodies, as described in “Materials and Methods.” (B) The bcr/ablphosphorylation status in LAMA84 and LAMA84R cell lysates treated with 1, 3, and 10 μM of STI571 or left untreated (control) was examined by immunoblotting using the monoclonal anti-phospho tyrosine antibody, as mentioned in “Materials and Methods.” The level of actin expression in the same lysates was detected as described above. (C) Immunocomplex kinase assay of bcr/abl in LAMA84 and LAMA84R. Cell lysates from both cell lines were immunoprecipitated with an anti-Abl antibody. The immunoprecipitates were left untreated (control) or treated with 3 μM STI571 and then subjected to an in vitro kinase assay as described under “Materials and Methods.” The upper panel shows the bcr/abl autophosphorylation level. The bcr/abl kinase activity was determined as GST (negative control) and GST-CH1 Shc phosphorylation (middle panel). Total cell lysates were subjected to Western blot analysis using anti-actin antibody to demonstrate equal protein levels in the samples analyzed (lower panel).