Fig 2: P120 participates in the RAC1-mediated upregulation of NIS PM residency. (A) HA-NIS-TPC1 cells were transfected with either an empty vector (Empty) or GFP-RAC1-V12 or with either mock (siLUC control) or a specific siRNA against P120 (siP120), as indicated. (B) HA-NIS-TPC1 cells were transfected with either mock (siLUC control), a specific siRNA against P120 (siP120), or a specific siRNA against VAV2 (siVAV2), as indicated. In addition, endogenous RAC1 activation status upon either P120 or VAV2 knockdown was assessed by monitoring the active GTP-bound fraction of RAC1 (using the CRIB domain pull-down assay) and comparing it to total (input) RAC1 levels, using anti-RAC1 primary antibody by WB. NIS-PM levels (A,B) were assessed by surface protein biotinylation and WB as described in Figure 1, Figure 2 and Figure 4. The plotted values correspond to the means ± SEM of at least three independent assays. One-way ANOVA analysis detected significant differences between the treatments (F = 19.30 and p = 0.0024 for (A); F = 33.59 and p < 0.001 for (B)). Significant variations relative to the control conditions or among the different treatments (indicated by horizontal lines) were identified by Tukey’s posthoc tests (** p = 0.01; *** p = 0.001). (C) Representative traces of the iodide-induced YFP fluorescence decay of HA-NIS/HS-YFP-TPC1 cells treated as in (B) and analyzed as described in Figure 1. (D) Iodide influx rates were derived from the decay curves. Data are the means ± SEM of at least three independent assays. Significant variations were detected by one-way ANOVA (F = 27.48; p < 0.001) followed by Dunnett’s posthoc test, compared to control conditions (Ctrl) (* p = 0.05; ** p = 0.01; *** p < 0.001). The uncropped bolts are shown in Supplementary Materials File S1.

Fig 3: Endogenous localization of p120-subfamily catenins in primary neurons. Endogenous localization of (A) p120-catenin; (B) delta-catenin; (C) ARVCF-catenin; (D) p0071-catenin in primary rat hippocampal neurons at 1 DIV and 5 DIV. Relative fluorescent intensity in the soma growth cones, processes, and nucleus is shown below each example image for 1 DIV (left) and 5 DIV (right). Cells were co-stained with Phalloidin and DAPI to help visualize growth cones and nuclei for quantification, but for clarity these stains are not pictured in example images. Statistical significance was determined using a two-way Brown-Forsythe and Welch ANOVA tests with Dunnett’s T3 multiple comparisons post-hoc test. Significance was assigned at P < 0.05 for *, and P < 0.0001 for ****, and ns for non-significant. N = 15 neurons from three biological replicates for each catenin at each DIV. Graph values show mean and standard deviation.