Fig. 3

CACHD1 physically interacts with Wnt receptors LRP6 and FZD family members.

(A) (Left) Representative scatter plot of flow cytometry testing binding of FLAG-tagged CACHD1 prey protein to human FZD7-EGFP transiently transfected HEK293E cells detected by means of phycoerythrin (PE)-conjugated secondary antibody. (Right) Without (blue) or with (red) preincubation with antibody to Frizzled OMP-18R5; secondary only negative control (gray). n = 3; one-tailed paired t test (DF = 2, t = 9.53, *** P = 0.0054). (B) Dot plot of human (blue diamonds) or zebrafish (blue circles) Cachd1 or negative control CD200R (gray) prey protein binding (standardized as ΔM_PE (supplementary materials, materials and methods) to cells transiently transfected with EGFP fusion protein constructs indicated (transfections verified by means of antibody labeling are indicated in bold). Each dot indicates a single experiment; horizontal bars indicate the mean, and error bars indicate 95% confidence intervals. One way Welch test of means (Cachd1 prey versus CD200R prey, not assuming equal variances; F = 132.32, DF_num = 30.00, DF_denom = 34.67, P = 5.09 × 10^-28), post hoc pairwise t tests with non-pooled standard deviations, Benjamini-Hochberg correction for multiple testing. Only statistically significant differences between Cachd1 and CD200R prey for individual transfections are presented here for clarity; ** 0.05 ≥ P > 0.01, *** 0.01 ≥ P > 0.005, **** P ≤ 0.005. (C) SPR-based determination of K_D (left) for mouse CACHD1_ECD analyte binding to immobilized mouse FZD5_CRD (cyan) or human LRP6_P3E3P4E4 (3-4, red), and normalized response curves (right) for different CACHD1_ECD:FZD_CRD interactions. RU, response units. (D) TOPFlash responses of HEK293 cells to WNT3A treatment after transfection with a control plasmid (white) or plasmids containing full length rodent Cachd1 (cyan), Cachd1 extracellular domain only (blue; ΔTM-C), or Cachd1 transmembrane and intracellular domains only (gray; ΔN). Mean responses are shown (n = 3 experiments; black dots indicate mean of quadruple technical replicates in each experiment); error bars idicate 95% confidence intervals. One way Welch test of means (not assuming equal variances; F = 13.202, DF_num = 3.00, DF_denom = 4.19, P = 0.014), post hoc pairwise t tests with non-pooled standard deviations, Benjamini-Hochberg correction for multiple testing. ns, P > 0.1, ** 0.05 ≥ P > 0.01.