Fig. 5.

Kuz promotes Robo cleavage in vitro. UASroboGFP with or without UASkuzHA were transfected into Drosophila S2R+cells. (A) Western blotting of separated proteins harvested from the media with antibodies to a N-terminal epitope of Robo reveals a significant increase in the amount of Robo ectodomain in the media of cells co-transfected with UASkuzHA compared with that of cells with no ectopic expression of kuz (lanes 1, 4 and 5). Co-transfection of UASrobo and UASkuz^Dmetallo did not increase the amount of Robo ectodomain detected in the media (lanes 1, 2 and 3). A lane between the first and second lane shown was excised from the media blot. Western blotting of the total lysates with antibodies directed against GFP, HA and tubulin show the relative levels of RoboGFP, KuzHA or Kuz^Δmetallo, and tubulin, respectively. (B) Western blotting of protein harvested from the media of cells transfected with UASHAroboMyc with or without UASkuzHA using an antibody directed against HA. (C) Western blots of proteins harvested from dsRNA-treated S2 cells. Cells expressing UASroboGFP, UASkuzHA, or both were treated with kuz dsRNA. The level of Robo ectodomain detected in the media from cells expressing both robo and kuz is lower in cells treated with kuz dsRNA (lanes 3 and 4). Treatment with kuz dsRNA also reduces the amount of Robo ectodomain detected in the media of cells with no transfected kuz (lanes 1 and 2). Western blotting of the total lysates with antibodies directed against Robo, HA and tubulin show the relative levels of RoboGFP, KuzHA and tubulin, respectively.