Supplementary Figure 5: Status of canonical WNT/β-catenin and MAPK pathways and expression levels of Fzd and Ryk mRNAs and ROR2 in Pkd2^+/+ and Pkd2^−/− MEFs.

Wild type or Pkd2-null MEFs were stimulated for 20 min with WNT9B (500 ng ml⁻¹) and phosphorylation and total levels of β-catenin (a), P38 MAPK (b) or LRP6 (c) were determined by immunoblotting. Experiments were done once. (d) Expression levels of DVL1 and DVL2 in Pkd1^−/−, Pkd2^−/− cells, and wild type MEFs derived from littermate control animals (Pkd1^+/+or Pkd2^+/+). Experiments were done 3 times. (e) Relative expression of Fzd1, Fzd2, Fzd6, Fzd7, Fzd8, and Ryk genes in Pkd2^+/+ and Pkd2^−/− cells determined by real time RT-PCR. Experiments were done three times in triplicates and data are shown as mean ± s.e.m. (f) Expression levels of ROR2 in both cell types determined by immunoblotting. Experiment was done once. (g) Status of canonical Wnt pathway in Pkd2^+/+ and Pkd2^−/− cells using a TCF-based transcription assay (TOP-Flash assay). (h) Suppression of canonical Wnt pathway activity by ZNRF3 in wild type (Pkd2^+/+) MEFs. (i) WNT9B did not activate the canonical Wnt pathway in wild type MEFs. Data from one representative out of three independent experiments all done in sextuplicates are shown in (g–i). Statistical analysis was performed using one-way ANOVA followed by Neuman-Keuls post hoc test. N.S means non-significant, ^∗∗∗ indicates P < 0.001. Data are shown as mean ± s.e.m. Statistics source data for all three independent experiments are available in Supplementary Table 3. Uncropped original scans of blots are shown in Supplementary Fig. 8.