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. 2015 Jul 15;29(14):1493–1506. doi: 10.1101/gad.264515.115

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© 2015 Cai et al.; Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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Figure 3. — Physical interactions among APC, Sav1, and Lats1 and their regulation by GSK-3β. (A–C) APC interacts with Sav1 and Lats1 in HEK293 cells. (A,B) Co-IP between Flag-APC and HA-Sav1 or Myc-Lats1. (C) Co-IP between endogenous APC and Lats1 and between endogenous APC and HA-Sav1. HA-Sav1 was used for co-IP analysis because endogenous Sav1 expression is very low in HEK293 cells. (D) Overexpression of APC enhanced Sav1–Lats1 interactions. (E) siRNA knockdown of endogenous APC decreased Sav1–Lats1 interactions. (F) HEK293 (control) and HEK293 with CRISPR–Cas9-engineered APC knockout (APCKO) were analyzed for co-IP between Sav1 and endogenous Lats1, showing reduced Sav1–Lats1 interaction in the APC knockout cells. (G) Western blot analysis of HEK293 cells after 6-bromoindirubin-3′-oxime (BIO) treatment for the indicated times. (H) Co-IP analysis of β-Trcp–YAP interactions. Note the reduction of β-Trcp–YAP interactions in HEK293 cells treated with BIO for 1 h. (I–K) similar to H except that APC–Sav1 (I), APC–Lats1 (J), and Sav1–Lats1 (K) interactions were analyzed.