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Fig. 3. Src-kinases phosphorylate ARAP3 at sites including Y1399 and/or Y1404 in cells and can phosphorylate ARAP3 in vitro. (A) ARAP3 (left panels) or FLAG-ARAP3 (right panels) were expressed with Lyn, Src or alone (none) in 293T cells. Cell lysates were immunoprecipitated (IP) with an ARAP3 antibody and analysed by SDS-PAGE and western blotting (WB) with phosphotyrosine (pY), ARAP3, Src or Lyn antibodies. (B) FLAG-ARAP3 (ARAP3 WT) and FLAG-ARAP3 with tyrosines 1399 and 1404 substituted for phenylalanines (Y1399/1404F) were expressed (+) with Lyn (L) or Src (S) in 293T cells. Cell lysates were immunoprecipitated (IP) with a FLAG antibody and analysed as in A with anti-pY and anti-ARAP3 antibodies. (C) FLAG-ARAP3 expressed in 293T cells and purified with a FLAG antibody was incubated with the indicated concentrations of purified Src kinase domain (upper panels) or with increasing amounts of immunopurified baculovirally expressed Lyn (lower panels) in in vitro kinase buffer containing ATP. Samples were analysed by SDS-PAGE and immunoblotting with the indicated antibodies.
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