1. Daple is required for activation of Gαi3, Rac1, and AKT signals and in the antagonistic inhibition of β-catenin–dependent Wnt signals downstream of EGF/EGFR.
Daple is required for activation of Gαi3, Rac1, and AKT signals and in the antagonistic inhibition of β-catenin–dependent Wnt signals downstream of EGF/EGFR. (A) Immunoprecipitation assay assessing Gαi3 activation after EGF stimulation in control (shLuc) and Daple-depleted (shDaple) HeLa cells. Western blot for active Gαi3 (Gαi3-GTP) and total Gαi3. Quantification of blots is shown in fig. S3. (B) Whole-cell lysates of EGF-stimulated HeLa cells in (A) were analyzed for phosphorylated AKT (pSer473), Daple, β-catenin, and actin (loading control). (C) Bar graphs display quantification of phosphorylated/total AKT. Data are means ± SD; n = 3 experiments. (D) Rac1 activity assay in lysates of HeLa cells described in (A) upon EGF stimulation. Data are means ± SD; n = 3. (E and F) Whole-cell lysates of EGF-stimulated HeLa cells expressing Daple-WT or Daple-F1675A (FA) mutant were analyzed for AKT phosphorylation, as described in (B). (G) Bar graphs display quantification as a ratio of phosphorylated to total AKT. Data are means ± SD; n = 3. (H and I) Rac1 activity upon EGF stimulation, assessed in lysates from HeLa cells described in (F). Data are means ± SD; n = 3. (J and K) Quantitative polymerase chain reaction (qPCR) analysis assessing EPCAM (J) and other Wnt target genes (K) induced by EGF in HeLa cells described in (A). Data are means ± SD, fold change in RNA after EGF stimulation; n = 3 experiments. n.s., not significant.
Nicolas Aznar et al., Sci. Signal. 2018;11:eaao4220
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