(A–C) Protein extracts from embryos expressing Robo1-Myc and various HA-tagged receptors in all neurons were immunoprecipitated with anti-Myc antibodies and analyzed by western blot. Immunoprecipitates were probed with anti-HA (top blots) and total lysates were compared for HA expression and Myc expression to ensure that equal inputs were analyzed. Representative western blots from multiple experiments are shown. (A) Robo1-Myc binds to HARobo1, HARobo2, and HARobo3 but not to a HA-tagged Fra receptor (two exposures are shown). Total lysate blots reveal comparable loading with the exception of the Fra negative control in which there is substantially more HA-tagged receptor. (B) Robo1-Myc binds efficiently to HARobo2, HARobo2∆Ig1, and the HARobo1Robo2(IG1-2) chimera but not to the reciprocal chimera that has Ig1 and Ig2 domains from Robo1 (asterisk). (C) Deletion of either Robo2 Ig1 or Ig2 alone does not substantially affect Robo1 binding, while deleting both domains results in reduced binding (asterisk). See Figure 11—figure supplement 1 for an additional example. (D) Cell lysates of S2R+ cells separately transfected for Robo1-Myc or HA-tagged Robo2 variants were mixed, immunoprecipitated with anti-Myc, and analyzed by western blot. In this assay, Robo1-Myc binds efficiently to HARobo2, HARobo2∆Ig1, and HARobo2∆Ig2, and less well to HARobo2∆Ig1+2 (asterisks). In lanes 1–4, cells were untreated; in lanes 5–8, cells were treated with Slit-conditioned media before lysing. We note that in addition to detection of the predicted full-length Robo2 receptor with anti-HA, we also routinely detect a smaller ∼80 kD fragment that corresponds to an extracellular domain cleavage product. The size of this fragment is shifted to predictably smaller sizes when Ig1, Ig2, or both Ig1 and Ig2 are deleted. We do not currently know, whether this cleavage event is required for Robo2 function in any context, since we have not been able to generate an uncleavable version of the receptor.