SWAP-70 is phosphorylated by Syk. (A) SWAP-70 kinase purification scheme with the resulting fractions and the respective active peak fractions indicated; FT indicates flow through. (B) Kinase reactions performed with 3 different amounts of recombinant SWAP-70 as indicated, and Fraction V of the kinase preparation. Autoradiograph of the gel showing phosphorylated SWAP-70 (at ca. 70 kDa) and autophosphorylated kinase (at ca. 75 kDa). (C) Silver-stained SDS-polyacryl amide gel showing the pooled activity peak fraction IV (IV), and subsequent fractions as eluted from the heparin Sepharose column (Fraction V, No. 18 to 27), and the heparin Sepharose flow-through (FT), besides a mass marker (M). Kinase activity of the individual fractions is indicated below. (D) IB of consecutive fractions from steps II, III, and V from the kinase purification. Kinase activity of the individual fractions is indicated below. The blot was probed with anti-SYK antibody, and the SYK protein signal is indicated. (E) Anti-SYK IB of an eluate from anti–SWAP-70 IP (right lane) and of 2 high-salt eluates (300 and 600mM ammonium sulfate) obtained from a SWAP-70 affinity column, onto which cytoplasmic extract from activated B cells was loaded. The specific SYK protein signal is indicated.