Analysis of Lyn activity and protein level during GA-mediated apoptosis of B-CLL cells. Unmutated CLL/ZAP⁺ (U-CLL/ZAP⁺) and mutated CLL/ZAP⁻ (M-CLL/ZAP⁻) cells were cultured for the indicated times, in the presence of 0.1 μM GA. (A) After GA treatment, U-CLL/ZAP⁺ and M-CLL/ZAP⁻ cells were lysed and analyzed by immunostaining with antibodies raised against PARP. Blots were stripped and reprobed with anti–β-actin antibody as loading control. (B) After GA treatment, U-CLL/ZAP⁺ and M-CLL/ZAP⁻ cells were lysed and analyzed by immunostaining with antibody raised against phospho-Tyr (pY). Molecular mass of protein standards are indicated in the middle. Blots were stripped and reprobed with anti–β-actin antibody as loading control. (C,D) After GA treatment, U-CLL/ZAP⁺ and M-CLL/ZAP⁻ cells were lysed by sonication in an isotonic buffer and subjected to differential centrifugation to separate cytosolic (C) and microsomal (D) fractions. Comparable aliquots were assayed for in vitro Lyn activity on Src-specific peptide substrate cdc2(6-20) and by Western blotting for Lyn. Lyn-specific activity is calculated as ratio of Lyn activity (bar graphs) over densitometric values of Western blot analysis for Lyn (panel below bar graphs) by standardizing the ratios of each control to the value of 100. All calculated SD values are less than 10%. Data are representative of experiments performed in triplicate on samples from each of 16 B-CLL patients.