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. 2017 Nov 6;216(11):3767–3783. doi: 10.1083/jcb.201612006

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© 2017 Wood et al.

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Figure 6. — αCat^KKR<3A mutant shows reduced recruitment to exogenous PIP₃. (A) PIP₃ apical membrane integration and recruitment assay; mCherry αCat (red); BODIPY–PIP₃ (green) with quantification of total colocalized surface. Arrows show colocalized surfaces as yellow (top) and purple (bottom) when reconstructed as a volume rendering. (B) BODIPY–PIP₃ did not alter expression of αCat (quantification not shown; n = 3). (C) Quantification of colocalization volumes between αCat constructs and PIP₃ (n = 45 from three BRs). Significance by unpaired t test: ***, P < 0.0005; ****, P < 0.0001. Error bars reflect the SD of the mean. (D) Surface renderings of colocalization volumes (purple) relative to BODIPY–PIP₃ integration (green). Slice shows en face view of Z-stack; arrows show colocalization (0.2-µm steps). (E) E-cadherin was not recruited to ectopic BODIPY–PIP₃ integrations, suggesting that this is a unique feature of extrajunctional αCat. Samples were fixed after live-cell imaging, so integration of BODIPY–PIP₃ was prolonged relative to Z-stacks in A and D. Bars, 20 µm.