Fig. 7. Src42A/E-cad/Arm ternary complex formation and Src-dependent Arm tyrosine-phosphorylation. (A) Arm/Src42A signals in E-cad immunoprecipitates of untransfected and Src42A/arm-transfected S2 cells. (B) Comparison of Arm/Src42A signals in cytosolic and membranous anti-Fas3 and anti-Arm immunoprecipitates of wild-type embryos. Identical amounts of membrane and cytosolic Arm were precipitated and examined. (C) Immunoprecipitation of membranous and cytosolic fractions of wild-type, Src42A[KR], Src42A[WT] and Src42A[YF] embryos. Same volumes of membrane and cytosolic fractions were analyzed. (a-d) Western blots of fractionated extracts. Tubulin is used as a control. (e-g) Arm, E-cad and Src42A blots of Anti-Arm antibody immunoprecipitates. (h) Src42A blots of anti-E-cad-antibody immunoprecipitates. (i,j) Arm blots of the supernatant of anti-Src42A antibody immunoprecipitates with (j) or without (i) anti-pTyr-antibody treatment. (D) Pull-down assay. Src42A was dissected as shown in the lower margin and expressed as MBP fusions. Y indicates the autophosphorylation site. GST-Arm-repeat signals were detected only in lanes 6 and 8, indicating Arm repeats to bind to the 14 amino acid, autophosphorylation-site-containing Src42A[Kinase] fragment. Lane 10 may indicate that the kinase domain other than the autophosphorylation peptide is unrelated to Arm/Src42A interactions. Input protein signals are shown in lower half as anti-MBP antibody signals. (E) Src dependency of Arm tyrosine phosphorylation. Western blots of extracts from S2 cells transfected with various combinations of metallothionein (MT)-GAL4 and UAS-arm, and dsRNAs indicated are shown (a-f). Signals for whole cell (d) and E-cad-free (e) tyrosine-phosphorylated Arm significantly reduced by Src42A RNAi (compare lane 2 with lane 5).