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. 2005 Jul 4;202(1):181–192. doi: 10.1084/jem.20050449

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Copyright © 2005, The Rockefeller University Press

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Figure 8. — Fyn and SAP are required for 2B4-mediated tyrosine phosphorylation signals. (A) Overall tyrosine phosphorylation. IL-2– expanded splenic NK cells from wild-type, SAP ⁻, and Fyn ⁻ ^/ ⁻ mice were stimulated (+) or not (−) for 5 min with biotinylated anti-2B4 antibodies and avidin. Protein tyrosine phosphorylation was examined by immunoblotting of total cell lysates with anti-P.tyr antibody (top). The abundance of 2B4 in the lysates was verified by reprobing the membrane with anti-2B4 antibodies (bottom). (B) Tyrosine phosphorylation of SHIP and 2B4. Experiment same as (A). Tyrosine phosphorylation of 2B4 (top) and SHIP (middle) was analyzed by probing anti-2B4 and anti-SHIP antibody immunoprecipitates (I.P.) with anti-P.tyr antibody. The abundance of 2B4 and SHIP was verified by reprobing the membranes with anti-2B4 and anti-SHIP antibodies, respectively. (C) Expression of SAP and Fyn in NK cells from wild-type, SAP ⁻, and Fyn ⁻ ^/ ⁻ mice. Experiment same as (A) and (B). The abundance of SAP and Fyn was verified in total cell lysates by immunoblotting with anti-SAP (top) and Fyn (bottom) antibodies.