NaChl inhibits Bcr-Abl and c-Abl autophosphorylation in Ph⁺ cells. (A) Immunoblot-based determination of Abl expression and phosphorylation status. Cells were treated with NaChl (10 μg/mL) for 30 minutes, harvested, and lysed, and equivalent amount of lysates were separated by SDS-PAGE and electrotransferred. The filters were probed with anti–phospho-c-Abl (Tyr 245) (top panels) or anti–c-Abl antibody (middle panels). β-Actin was used as loading control (bottom panels). (B) In vitro kinase assay of fused (Bcr-Abl) and unfused Abl in the presence (T) or absence (NT) of NaChl (50 ng/mL, 30 minutes). (C) HSG and HSC-2 cells do not express Bcr-Abl. Cells were harvested and lysed, and equivalent amount of lysates were separated by SDS-PAGE and electrotransferred. The filters were probed with indicated antibodies. (D) Flow cytometric determination of Abl phosphorylation status. Indicated cells were left untreated (NT) or incubated with NaChl (10 μg/mL) for 30 minutes (T), fixed, permeabilized, and stained with rabbit anti–phospho-c-Abl (Tyr 245) antibody. Dotted line indicates staining with normal rabbit sera; solid line, staining with anti–phospho-c-Abl antibody. (E) Flow cytometric determination of intracellular Abl protein expression status. Cells were fixed, permeabilized, and stained with rabbit anti–c-Abl antibody. Dotted line indicates staining with normal rabbit sera; solid line, staining with anti–c-Abl antibody.