SWAP-70 is modified by tyrosine phosphorylation. (A) SWAP-70 is posttranslationally modified after LPS stimulation. Cytoplasmic extracts from stimulated and unstimulated B cells were analyzed by 2D Western blotting with anti–SWAP-70. Isoelectric focusing was performed between pH 4 and 7. The unmodified form of SWAP-70 has an approximate pI of 5.85 and is indicated, after activation several modified forms of SWAP-70 appear. (B) SWAP-70 is phosphorylated. LPS-stimulated B cells were grown in the presence of γ-³²P-ATP and then SWAP-70 underwent IP from cytoplasmic extracts. The presence of SWAP-70 in precipitates was confirmed by SDS-PAGE of the precipitate and subsequent silver staining. On the silver stain, a band at 70 kDa corresponding to SWAP-70 can be seen, a corresponding band is found in the autoradiograph. (C) Stimulation of B cells leads to tyrosine phosphorylation of SWAP-70. IPs were made from nuclear (N) and cytoplasmic (C) extracts of stimulated and unstimulated cells with antiphospho-Tyr (α-P-Tyr). The resulting precipitates underwent IB with anti–SWAP-70 to determine in which samples SWAP-70 is Tyr phosphorylated. SWAP-70 was precipitated with antiphospho-Tyr from stimulated cell nuclear and cytoplasmic extracts but not unstimulated cells. Shown are representative images from experiments performed at least 3 times.