Fig. 4. Yki phosphorylation and 14-3-3 binding. (A) Western blots (4-15% PAGE) of samples from S2 cells co-transfected with the indicated proteins. Bottom two panels show blot on material precipitated with anti-V5 beads, upper two panels shows input; GFP:V5 (control protein) and 14-3-3:V5 proteins have similar mobility. (B) Western blots (using anti-V5 epitope) on lysates of S2 cells co-transfected to express the indicated proteins. Transfection of Wts, Hpo and Sav promotes phosphorylation of Yki and Yki-S168A, as revealed both on conventional SDS-PAGE (top panels) and Phos-Tag gels (30 µM Phos-Tag, bottom panels); however, a distinct mobility isoform (arrow), representing partially phosphorylated Yki, is observed with Yki-S168A mutant but not wild-type Yki on Phos-tag gels. Transfer of heavily phosphorylated proteins from Phos-Tag gels can be poor, hence they may be under-represented on these blots. (C) Western blots of lysates of wing imaginal discs from animals expressing Yki:GFP (lane 1) or Yki-S168A:GFP (lane 2) under sd-Gal4 control. Upper panel shows conventional SDS-PAGE, lower panel shows a Phos-tag gel (60 µM Phos-Tag). (D) Western blot on lysates of S2 cells co-transfected to express the indicated proteins, run on a Phos-tag gel (60 µM Phos-Tag). Yki-N is an N terminal fragment of Yki comprising the first 240 amino acids.