Aberrant phosphorylation state of Lyn in B-CLL. (A) Schematic representation of domain structure and functional properties of Lyn along with the 2 phosphorylation sites recognized by the specific antibodies. (B) Whole B-cell lysates (lanes 1 and 4), microsomes (lanes 2 and 5), and cytosol (lanes 3 and 6) were assayed by Western blot analysis with anti-LDH (cytosolic marker), anticalnexin (microsomal marker), antilamin (nuclear marker), and antiaconitase (mitochondrial marker) antibodies. Western blots are representative of samples from 5 normal donors (left, lanes 1-3), and of those from 10 CLL patients (right, lanes 4-6) are shown. (C) Whole B-cell lysates (lanes 1 and 4), microsomes (lanes 2 and 5), and cytosol (lanes 3 and 6) from one normal donor (lanes 1-3) and from CLL patient 15 (lanes 4-6) were analyzed by immunoblotting with anti-pY_A, anti-pY_T and, after stripping, reprobed with anti-Lyn antibody. Molecular weight (kDa) corresponding to p53 and p56 isoforms of Lyn are indicated in the middle. (D) Densitometric analysis (arbitrary units) of anti-pY_A, anti-pY_T, and anti-Lyn bands of whole cell lysates (lanes 1 and 4), microsomes (lanes 2 and 5), and cytosol (lanes 3 and 6) from 5 normal (lanes 1-3) and 40 B-CLL samples (lanes 4-6) is shown. Data are mean plus or minus SD from 3 separate experiments. Whole cell lysates, microsomes, and cytosol were prepared as detailed in “Methods.”