Figure 6: EEF1A depletion increases E-cadherin levels at junctions without strengthening cell adhesion.

Keratinocytes grown in low-calcium medium were depleted of EFF1A before junctions were either induced for 30 min (a–c,e,f) or aggregation assays (d) and actin recruitment (g) performed. (a) E-cadherin staining images were collected and shown as representative inverted images. (b) E-cadherin levels at junctions were quantified as thresholded levels (% area) and normalized to controls (non-targeting oligos). (c) Total protein levels of cadherin and catenins following RNAi treatment. (d) Cells were allowed to aggregate in suspension for 2 h, followed by mechanical trituration (see Supplementary Fig. 7a). Disaggregate sizes were measured and expressed relative to controls. (e,f) Before fixation, keratinocytes were pre-extracted with TX-100-containing buffer and the insoluble pool of E-cadherin at junctions was quantified (f). (g) F-actin recruitment to clustered E-cadherin receptors around latex beads was assessed by phalloidin staining. (h–j) Dynamics of junctional actin in mature junctions was assessed. Following bleaching of selected areas at junctions, fluorescence recovery was recorded (h). Quantification of final fluorescence recovery levels (i) and the half-time required to reach the plateau in each sample (j) are shown. (k) Pull downs using GST or GST-EEF1A were performed and co-precipitated endogenous DIAPH2, VAV2 or FILB detected. (l,m) Similar experiments as in k were performed following expression of flag-Diaph2 wild-type (mouse mDia3, l) or activated Diaph3 (mouse mDia2, ΔGBD) and mutated at the predicted EEF1A-binding site (ΔGBDΔDAD^mEBS; m) Molecular weight markers are shown on the right of each panel. Blots and immunofluorescence images are representative of independent replicates. Fusion proteins are detected with Amido Black staining. Scale bars, 20 μm (a,e); 10 μm (g); 2 μm (h). Error bars represent standard error of the means of independent replicates (d (oligo1), f,g, N=3; b,d (oligo2) N=2; i,j, control N=8, EEF1A N=13). Statistical analyses were performed with two-way Anova (b,f) or Student t-test (d,g,i,j). @ P=0.01; *P=0.007; **P=0.0002; ***P=0.002.