Figure 3. Molecular characterization of the influence of Fj on Fat-Ds binding.

Histograms show the results (averages of from two to four replicate experiments) of binding of AP-tagged Ds proteins, with bound AP activity expressed as milli-OD/min. Error bars indicate standard deviation. A) In vitro Fat-Ds binding assays, using conditioned medium from cells expressing Ds1-10:AP (2400 mOD/min) and beads loaded with Fat1-10:FLAG from S2 cells, Fat1-10:FLAG from S2 cells co-transfected to express Fj, or conditioned medium from S2 cells transfected with empty vector, as indicated. Where indicated, beads were phosphorylated in vitro with Fj or treated with phosphatase. For simplicity of display, the “−” samples here display averages from the mock treated (no enzyme) kinase and phosphatase experiments. Inset shows the results of Western blotting (anti-FLAG) on Fat1-10:FLAG from S2 cells without or with (+Fj) Fj co-expression to show that the amounts of Fat1-10 are similar. B) In vitro Fat-Ds binding assays, using conditioned medium from cells expressing Ds1-10:AP (2400 mOD/min) and beads loaded with Fat1-10:FLAG from S2 cells with or without Fj co-transfection, or conditioned medium from S2 cells, as indicated. Where indicated by superscripts, Fat1-10:FLAG with point mutations in the Fj site in Cadherin domain 3 were used. C) Cell based binding assays, using conditioned medium from cells expressing full length DS:AP or Ds1-10:AP (1200 mOD/min), with or without Fj co-expression, as indicated. Cells were transfected to express full length Fat, Fat and Fj together, Fat and kinase-dead Fj (Fj^GGG), or empty vector (S2). D) In vitro Fat-Ds binding assays, using conditioned medium from cells expressing Ds1-10:AP (2400 mOD/min) with or without Fj co-expression, or as a control Fc:AP, as indicated, and beads loaded with Fat1-10:FLAG from S2 cells co-transfected to express Fj.