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. 2014 Nov 18;5:5482. doi: 10.1038/ncomms6482

Figure 7. Polycystin complex containing cleaved PC1 traffics to cilia through a Rabep1/GGA1/Arl3-dependent mechanism.

Figure 7

(a) Co-immunoprecipitation of endogenous PC1 with Rabep1, GGA1 and Arl3 from CD cells. CD cells with shRNA-mediated Pkd1 knockdown were used as a negative control. The larger areas of the blots are shown in Supplementary Fig. 3d. (b) Co-immunoprecipitation of C-terminal Myc-tagged PC1 with Rabep1, GGA1 and Arl3 from Pkd1MYC/MYC embryo lysates (MYC). Pkd1ΔCMYC/ΔCMYC knockout embryos that expresses truncated PC1 lacking the C-terminal 257 amino acids (ΔCMYC) and untagged wild-type (WT) embryos were used as negative controls. (c) Wild-type (WT) or Pkd2−/− MEF cell lysates were immunoprecipitated with anti-PC1 (α-CC), and the IP products were analysed with the antibodies as indicated. (d) Knockdown of Rabep1, GGA1 or Arl3 in CD cells abolishes ciliary localization of endogenous PC1 (left panels) and PC2 (right panels). The white line in the XY scan indicates the path of the XZ scan. For knockdown of each target protein, three shRNAs for Rabep1, four shRNAs for GGA1 and two shRNAs for Arl3 were used, and each lentiviral shRNAs similarly inhibit the expression of the target proteins by ~90% (Supplementary Fig. 3a–c). Scale bar, 10 μm. (e) Quantification of PC1 and PC2 ciliary localization in CD cells expressing various shRNAs in (d) was performed from four independent experiments and presented as the mean±s.e.m.; ***P<0.001. (f) Endogenous PC1 were immunoprecipitated with anti-PC1 (α-CC) from CD cells expressing the shRNA as indicated and analysed with various antibodies as indicated. Note that acquisitions of EndoH resistance of PC1 and PC2 were not affected by any of the shRNAs. The larger areas of the blots are shown in Supplementary Fig. 3e. (g) Co-immunoprecipitation of native GGA1 and Arl3 in CD cells with Rabep1 knockdown. The specificity of the GGA1–Arl3 interaction was validated using CD cells with GGA1 knockdown (Supplementary Fig. 4d). (h) Co-immunoprecipitation of GGA1 with Rabep1 and Arl3 in both wild-type (WT) and Pkd1−/− MEFs.