Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

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

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 2017 Sun et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).

PMC Copyright notice

Figure 4. — Scrib mediates reverse signaling of Sema4A. (a) Workflow for the identification of proteins interacting with the intracellular portion (IC) of class 4 semaphorins by mass spectrometry. For details, see Materials and methods. (b) MIA PaCa-2 cells transfected with the indicated siRNAs were examined in transwell migration assays in the absence or presence of 150 nM ecPlxnB1. The ecPlxnB1-induced effect on cells transfected with siRNA directed against the gene of interest is normalized to the ecPlxnB1-induced effect on cells transfected with control siRNA (percentage). Genes encoding for proteins identified as potential Sema4A-interacting partners by the GST pulldown approach are colored in red. Error bars represent the mean ± SD. (c) HEK293T cells were transfected with plasmids encoding HA-tagged Scrib (HA-Scrib) alone or together with V5-tagged Sema4A (V5-Sema4A). After serum starvation and stimulation with or without ecPlxnB1, cells were lysed, and proteins were immunoprecipitated (IP) using anti-V5 or anti-HA antibodies coupled to protein A/G sepharose. Bound proteins were then separated and visualized using anti-HA or anti-V5 antibodies (as indicated). (d) MIA PaCa-2, CaD2, or T3M4 cells were serum starved and stimulated as indicated. Sema4A or Scrib were immunoprecipitated using the respective antibodies (IP). Protein complexes were visualized by Western blotting (immunblotting [IB]). (e) Mature BMDCs were generated from wild-type mice (WT) or Sema4A knockout mice (Sema4A KO). Cells were stimulated with or without 150 nM ecPlxnB1 and lysed, and protein complexes were immunoprecipitated using anti Scrib antibodies. Proteins were then immunoblotted using specific antibodies as indicated. (f) Schematic representation of GFP-tagged Scrib constructs used in this study. (g) HEK293T cells were transfected with V5-tagged Sema4A and constructs encoding GFP-tagged wild-type or deletion mutants of Scrib (as indicated). Protein complexes were immunoprecipitated in the presence of 150 nM ecPlxnB1 using anti-V5 antibody and visualized using the indicated antibodies. Error bars represent means ± SD. *, P < 0.05.