NOS1AP interacts with β-Pix through an interaction with Scrib. A, B, HEK293T cells were transfected with cDNA constructs as indicated. The resulting lysates were immunoprecipitated (I. P.) with anti-GFP antibody (A) and probed as indicated. B, Lysate controls for A. C, D, HEK293T cells were transfected with cDNA constructs as indicated. The resulting lysates were immunoprecipitated with anti-GFP antibody (C) and probed as indicated. D, Lysate controls for C. E, HEK293T cells were transfected with cDNA constructs as indicated. The resulting lysates were immunoprecipitated with anti-GFP antibody and probed as indicated. F, Left panel, Stable Cell lines generated with a Scrib shRNA knockdown vector or control vector show reduction in Scrib levels (upper panel) compared to Git1 (middle panel) or tubulin (lower panel). Right panel, Control or Scrib knockdown cell lines were immunoprecipitated with preimmune (P.I.) or NOS1AP-specific antibodies as indicated and then probed with anti-Scrib (upper panel) or anti-Git1 (lower panel). G, Equal amounts of cleared lysate from the Scrib knockdown cell line or a control cell line were incubated with GST-PBD then probed with anti-Rac to determine the level of activated Rac (upper panel). Total Rac level (lower panel). H, Summary of the interactions outlined in A–E.