Figure 7.

Scrib and βPIX are required for Plexin-B1–Sema4A reverse signaling. (a) HEK293T cells transfected with V5-Sema4A, GFP-Scrib, and FLAG-βPIX were stimulated with or without 150 nM ecPlxnB1. Protein complexes were precipitated using anti-GFP antibodies and visualized with immunoblotting (IB). IP, immunoprecipitation. (b) MIA PaCa-2 cells were transfected with siRNAs as indicated, and cell migration in response to 150 nM ecPlxnB1 or 10% FBS in a transwell system was analyzed as described in Materials and methods (total n = 6 per condition). (c and d) THP1 cells were fully differentiated to DCs as described in Materials and methods. Cells were then stimulated with 150 nM ecPlxnB1 or 25 ng/ml CCL19 (as indicated), and Cdc42 activity (c) and cell migration (d) was analyzed by using a transwell system as described in Materials and methods (total n = 6 per condition). ns, not significant. CTRL, control. (e) MIA PaCa-2 cells were transfected with cDNAs encoding GFP or GFP-Scrib (amino acids 1224–1630), and cell migration in response to ecPlxnB1 was measured as described in Materials and methods. Shown are mean values ± SD from three independent experiments (total n = 8 per condition). Error bars represent means ± SD. **, P < 0.01; ***, P < 0.001.