BCR-ABL expression, tyrosine phosphorylation, and signaling in K562 and K562-R cells.

(A) BCR-ABL and protein tyrosine phosphorylation levels were analyzed by immunoblotting equal protein (30 μg) cell lysates. For analysis of BCR-ABL protein expression (top), cell lysates (300 μg) were immunoprecipitated with anti-bcr (2 μg) prior to immunoblotting with anti-abl^8E9. Relative migration of protein standards is shown at left. The most notable distinction between cell lines was expression of a 60-kDa tyrosine phosphoprotein (p60). (B) Northern blot analysis of BCR-ABL mRNA expression in K562 and K562-R cells. Total RNA (20 μg) from K562 and K562-R cells was blotted with a 1.3-kb BCR-ABL probe as described in “Materials and methods.” The extent of hybridization with probe is shown (top), and the migration of 28S and 18S RNA is shown at the bottom. (C) 30 μg protein from K562 and K562-R cells were immunoblotted with anti-abl (top), phosphoStat5 (p-Stat5), Stat5, phosphoMAPK (p-MAPK), or MAPK. CrkL phosphorylation (p-CrkL) was determined by immunoprecipitation from cell lysates (300 μg) and blotting with antiphosphotyrosine (4G10; Upstate Biotechnology Institute). CrkL levels were determined by stripping the membrane and reprobing with anti-CrkL.