Fig. 5. Drosophila Spry is phosphorylated and binds a substrate-trapping form of Csw. (A) Tyrosine phosphorylation of Spry. S2 cells expressing Breathless-FLAG were mock-treated (lanes 1, 3) or treated with 0.1 mM pervanadate, a tyrosine phosphatase inhibitor (lanes 2, 4) and endogenous Spry was immunoprecipitated from cell extracts and analyzed on immunoblots. (Lanes 1, 2) Immunoblot probed with anti-phosphotyrosine to detect phosphorylated Spry. (Lanes 3, 4) Immunoblot probed with anti-Spry to detect all Spry (70 kDa isoform, Spry70; doublet of 42 kDa isoforms, Spry42). There is tyrosine phosphorylation of Spry70 and a Spry42 isoform (lane 2). Similar results were obtained in three experiments. (B,C) Requirement of Spry Y201. (B) Section through eye of w; sev-GAL4/UAS-spry fly that expresses Spry under control of sev-GAL4 in developing eye. Ommatidia are disorganized and some (arrowheads) are missing photoreceptors. (C) Similar section of w; sev-GAL4/UAS-spry^Y201F that expresses Spry^Y201F under control of sev-GAL4. Ommatidia appear normal. (D) Binding of Spry to substrate-trapping Csw in S2 cells. Whole cell lysates were prepared from transfected S2 cells expressing Csw (lanes 1, 3) or Csw^C583S, a substrate-trapping form of the enzyme (lanes 2, 4). Aliquots of lysates were directly resolved by SDS-PAGE (lanes 1, 2) or first immunoprecipitated with anti-Csw (lanes 3, 4). Immunoblots were probed with anti-Spry or anti-Csw as indicated. More Spry co-immunoprecipated with Csw^C583S (lane 4) than with Csw (lane 3). Similar results were obtained in three experiments. (E) Binding of Spry to substrate-trapping Csw in imaginal discs. Eye-antennal discs dissected from third instar transgenic larvae expressing myr-Csw, myr-Csw^C583S or myr-Csw^G547E, a dominant-negative Csw that does not function as a substrate trap, were homogenized and directly resolved by SDS-PAGE (lanes 1-3) or immunoprecipitated with anti-Csw and then resolved by SDS-PAGE (lanes 4-6). Immunoblots were probed with anti-Spry or anti-Csw as indicated. Positions of Spry42 and Csw are shown; Spry70 is variably detected in eye disc lysates. More Spry42 co-immunoprecipitated with myristylated Csw^C583S than with myristylated Csw or Csw^G547E. A similar result obtained in a repeat experiment, except in this case Spry70 isoform predominated in co-immunoprecipitate.