Supplementary Figure 3: Expression levels of transfected proteins in CHO-K1 cells and biochemical characterization of TRPP2^Kv1.3 in HEK293T cells.

(a) CHO-K1 cells were transiently transfected with (pCDNA3, lane 1), wild-type HA-tagged PKD1 plus wild type TRPP2 (lane 2), wild type HA-PKD1 plus HA-tagged TRPP2^D511V (lane 3), wild type HA-PKD1 plus TRPP2^Kv1.3 (lane 4), wild type HA-PKD1 plus wild type TRPP2 plus HA-tagged ZNRF3 (lane 5), or HA-PKD1^D99I plus wild type TRPP2 (lane 6). PKD1 was immunoprecipitated from lysates pooled from three plates using mouse monoclonal 7e12 and blotted with the same antibody (upper panel). TRPP2 constructs were detected in straight lysates using goat α-TRPP2 (middle panel) and the blot was sequentially probed with α-HA to detect ZNRF3 (lower panel). (b) Results from one representative out of three independent experiments all done in sextuplicates showing the effectiveness of ZNRF3 in WNT3A-induced canonical signaling in CHO-K1 cells. Statistical significance was determined One Way ANOVA Neuman-Keuls posthoc test, t-test, ^∗∗∗ indicates P < 0.001. Data are shown as mean ± s.e.m. Statistics source data for three independent experiments are available in Supplementary Table 3. (c) TRPP2^Kv1.3 homodimerizes and interacts with wild type TRPP2. HEK293T cells were transiently co-transfected with HA-tagged TRPP2 (HA-TRPP2) and myc-tagged TRPC3 (TRPC3-myc, negative control, lane 1), HA-TRPP2 and TRPP2^Kv1.3-myc (lane 2), HA-TRPP2 and TRPP2-myc (positive control, lane 3), HA-TRPP2^Kv1.3 and TRPC3-myc (negative control, lane 4), HA-TRPP2^Kv1.3 and TRPP2^Kv1.3-myc (lane 5), or HA-TRPP2^Kv1.3 and TRPP2-myc (lane 6). HA-tagged proteins were immunoprecipitated with mouse α-HA and immunoblotted with rabbit α-myc (upper panel) or rabbit α-HA (lower panel). Expression levels of myc-tagged proteins are shown in middle panel. (d) TRPP2^Kv1.3 interacts with PKD1. HEK293T cells were transfected with indicated plasmids and F-PKD1 was immunoprecipitated with mouse monoclonal α-Flag. Imunocomplexes were immunoblotted with rabbit polyclonal α-myc (upper panel) or rabbit α-Flag (middle panel). Expression levels of TRPP2-myc or TRPP2^Kv1.3-myc are shown in lower panel. An irrelevant lane shown by a dotted line was deleted between lanes 3 and 4. Experiments were successfully repeated 3 times, except in (a), where experiments were done once. Uncropped original scans of blots are shown in Supplementary Fig. 8.