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. 2016 Feb 1;27(3):466–482. doi: 10.1091/mbc.E15-09-0632

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© 2016 Mills et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 7: — Truncated APC does not interact with the Miro/Milton complex. (A and B) Test for interaction between APC and Miro/Milton in colon cancer cell lines SW480 (APC1377), HT-29 (APC853/1555), and LIM1215 (APC WT) in situ using Duolink PLA. (A) Representative cell images (green dots are positive signals) were quantified in the graph (B). The interaction between APC and Miro/Milton is lost when APC is truncated (SW480 and HT-29 CRC cells) but remains intact when APC is full length (LIM1215 CRC cells). (C) SW480 cells expressing GFP, GFP-Miro-1, or GFP-Milton-2 were subjected to IP assays using anti-APC to pull down the respective GFP-tagged protein and anti-GFP to pull down APC. No bands were detected. Blots were spliced where indicated. (D) SW480 cell lysates were used for IP of endogenous protein; however, antibodies against APC were unable to pull down endogenous Miro-1 or Milton-2. Blots were spliced where indicated. (E) SW480 cells transiently expressing GFP-APC-WT and GFP-APC-1309 were subject to Duolink PLA where interactions between GFP-tagged APC sequences and Miro-1 were observed in situ by immunofluorescence microscopy. (F) Quantification of PLA signals revealed a significant positive signal above GFP background for GFP-APC-WT that was not observed for the GFP-APC mutant (***, p < 0.001; n.s., not significant). n > 125 per sample over two independent experiments. Box-and-whisker plot presented as a median (line), upper/lower quartile (box), and min/max (error bars with 95% CI). Statistical analysis by Mann-Whitney U-test with Bonferroni correction. Scale bars: 10 μm.