Dephosphorylation of Lyn's regulatory sites by CD45 and induction of their phosphorylation by BCR ligation. (A) 32P-orthophosphate–labeled WEHI-231 and CD45-deficient clone 10-5 cells were left unstimulated (–) or were stimulated (+) for 5 minutes with 20 μg/mL anti-IgM Ab, after which Lyn was immunoprecipitated from GEMs and subjected to SDS-PAGE analysis. 32P-labeled Lyn bands were excised from the PVDF membrane, hydrolyzed, and subjected to phosphoamino acid analysis. S indicates serine; T, threonine; and Y, tyrosine. (B) Cells were treated as in panel A, and the excised Lyn bands were treated with CNBr. The eluted proteins were subjected to SDS-PAGE in a 15% to 25% gradient gel, transferred to a PVDF membrane, and analyzed with a BAS 2000. The Lyn protein applied was assessed by Western blotting with anti-Lyn Ab. Y397 indicates the autophosphorylation site; Y508, negative regulatory site; and MW, molecular weight.
