Induction of tyrosine phosphorylation through VLA/β1-stimulation. (A) Induction of several proteins' tyrosine phosphorylation in peripheral lymphocytes from two normal controls (C 1 and 2) and 4 patients (PN 1, 2, 3, and 4) 8 weeks to 2.5 years after allo-BMT. Monocyte-depleted lymphocytes were incubated on plates coated using rabbit antimouse IgG with (+) or without (−) 4B4 (5 μg/mL) for 30 minutes, and then lysed. Whole cell lysate (50 μg/lane) was immediately subjected to Western blotting with the anti-phosphotyrosine MoAb. Autoradiography was carried out for 14 to 18 hours. Normal control samples were analyzed at the same time in each experiment, which constantly demonstrated similar tyrosine phosphorylation pattern to Ct1 and Ct2 (data not shown). Representative results at each stage after allo-BMT from different membranes are shown. (B) Whole thymocytes and immunodepleted thymocytes using anti-CD4 (19Thy) plus anti-CD8 (21Thy). Anti-CD45RA (2H4) or anti-CD45RO (UCHL1) antibody were incubated on plates coated with rabbit antimouse IgG (RbαM) or rabbit antimouse IgG plus anti-CD29 antibody (4B4; 5 μg/mL) for 30 minutes, lysed, and detected by anti-pTyr blot by the same method as above.