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. 2017 Jan 2;216(1):199–215. doi: 10.1083/jcb.201602002

Figure 6.

Figure 6.

Plexin-B1–Sema4A signaling induces internalization of Scrib. (a) T3M4 cells were incubated with or without 150 nM ecPlxnB1, and Scrib was visualized by immunostaining at the indicated time points after application of ecPlxnB1. Scrib localization was analyzed using ImageJ as described in Materials and methods. Time point 0 min: n = 41; 2.5 min: n = 61; 5 min: n = 54; 10 min: n = 53; 20 min: n = 26; 30 min: n = 45. (b) T3M4 cells stably transfected with control shRNA or with Sema4A shRNA alone or together with either shRNA-resistant wild-type Sema4A (S4A) or an shRNA-resistant Sema4A intracellular deletion mutant (S4AΔC) were incubated with 150 nM ecPlxnB1 for 30 min, and Scrib was visualized by immunostaining. Scrib localization was analyzed using ImageJ as described in Materials and methods. Bar graphs show mean values ± SD from at least 10 cells per condition. (c) Surface proteins of T3M4 cells transfected with control or Scrib were biotinylated. Cells were then incubated with or without 150 nM ecPlxnB1 for 30 min, surface proteins were precipitated using streptavidin agarose, and bound Sema4A was visualized by Western blotting using an anti-Sema4A antibody (pulldown; right). Band intensities were quantified from six independent Western blots (left). ns, not significant. CTRL, control; IB, immunoblotting. Error bars represent means ± SD. *, P < 0.05; ***, P < 0.001.