(A) Phylogenetic sequence analysis reveals a conserved motif in Daple similar to GIV's Gα-binding and activating (GBA) motif within an otherwise highly divergent C-terminal domain. Sequences of GIV …
Purified, recombinant GST-Gαi3 (WT and mutants) preloaded with GDP and immobilized on glutathione-agarose beads was incubated with purified His-Daple-CT (aa 1650–2028) as indicated. Resin-bound …
(A) Prediction of molecular contacts critical for the Daple-Gαi interaction. Homology-based model of Daple's GBA motif (Red) bound to Gαi3 (green = Switch II, blue = ras-like domain, yellow, …
(A, B) Daple competes for binding to Gαi3 with peptides/proteins that dock onto the switchII/α3 cleft of the G protein. (A) Purified, recombinant GST-Gαi3 preloaded with GDP and immobilized on …
(A) Daple's GBA motif and Gβγ subunits are predicted to dock onto an overlapping binding site on Gαi. Binding areas (in red) for Daple (left) or Gβγ (right) on Gαi (solid gray) were extracted from a …
(A) Daple and Gαi3 co-immunoprecipitate with FZD7R after Wnt5a stimulation. HeLa cells cotransfected with myc-Daple WT and HA-FZD7 were starved and stimulated with Wnt5a as in 3G. Equal aliquots of …
(A) A sequence homology-based cluster tree of vertebrate Frizzled receptors (FZDRs) is shown. The FZD (IUPHAR nomenclature) family roughly clusters into four distinct families based on sequence …
(A) HEK cells expressing HA-tagged FZD7R were starved for 24 hr (0% FBS) and stimulated with Wnt5a for 5 min as indicated prior to lysis. IP was carried out on lysates with anti-HA or control mouse …
(A) Dishevelled (Dvl)–Daple complexes are disrupted upon Wnt5a stimulation. HeLa cells cotransfected with myc-Daple-WT and Dvl were incubated in a 0.2% serum media overnight, and treated (+) or not …
(A) Equal aliquots of lysates from Cos7 cells expressing Dvl1 alone (lane 2), myc-Daple alone (lane 3), or coexpressing both (lanes 1, 4) were used as source of Daple and Dvl in GST pulldown assays …
(A–D) Daple does not activate Gi after Wnt3 stimulation. (A, B) Control (Luc shRNA) or Daple-depleted (Daple shRNA) HeLa cells were cotransfected with Gαi1-YFP, Gβ-CFP, and untagged Gγ, serum …
(A) Daple WT, but not FA triggers chemotactic migration towards Wnt5a. Daple-depleted HeLa cells (sh Daple 1) stably expressing Daple-WT or Daple-FA were analyzed for their ability to migrate …
(A) Daple-FA, but not Daple-WT inhibits 2-D cell migration. Confluent monolayers of HeLa cells transiently transfected with myc-Daple WT or FA (∼90% efficacy of transfection confirmed by …
(A) Daple's GBA motif is required for inhibition of cell transformation induced by oncogenic KRas. NIH3T3 cells stably expressing HA-KRas G12V alone or coexpressing HA-KRas G12V with myc-Daple-WT or …
(A) Compared to cells expressing Daple-WT, those expressing Daple-FA have higher mitotic index, as determined by nuclear localization of phosphorylated histone H3. HeLa cells expressing myc-Daple WT …
(A) Daple mRNA is downregulated in colorectal cancers. A meta-analysis was performed using all the available high-throughput microarray data from Genomic Spatial Event (GSE) database (see Figure …
Meta-analysis of Daple mRNA expression in colorectal cancer vs matched normal controls.
The publicly available GSE database, a system to store, retrieve, and analyze all types of high-throughput microarray data was used to compare the levels of expression of Daple mRNA in colorectal cancer vs matched normal controls. From left to right, the columns indicate the GSE series ID, the PMID number for the respective source manuscripts, total samples analyzed in each study, fold change in Daple mRNA observed, and the significance (p-value) of any changes observed. A meta-analysis combining the p-values from these studies was analyzed by Fisher's method and displayed as bar graphs in Figure 8A.
Meta-analysis of Daple mRNA expression in microsatellite unstable (MSI) vs stable (MSS) colorectal cancers.
The publicly available GSE database was used to compare the levels of expression of Daple mRNA in MSI vs MSS colorectal cancers. From left to right, the columns indicate the GSE series ID, the PMID number for the respective source manuscripts, total samples analyzed in each study, fold change in Daple mRNA observed, and the significance (p-value) of any changes observed. A meta-analysis combining the p-values from these studies was analyzed by Fisher's method and displayed as bar graphs in Figure 8C.
Daple expression in CTCs correlates with markers of EMT.
Expression of Daple, ZEB2, and LOXL3 mRNA were analyzed in CTCs immunoisolated from 50 patients with metastatic colorectal cancer. An analysis of the Pearson's correlation coefficient for each pair of genes shows that higher expression of Daple is significantly associated with higher expression of ZEB2 and LOXL3, two genes implicated in triggering EMT.
(A, B) Publicly available Kaiser Colon database was analyzed for Daple mRNA expression in adenocarcinomas of the colon (A) and rectum (B) and their respective normal controls. Daple mRNA expression …