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. Author manuscript; available in PMC: 2009 Nov 1.
Published in final edited form as: Nat Cell Biol. 2009 Mar 29;11(5):545–556. doi: 10.1038/ncb1861

Figure 3. PKD1 phosphorylates SSH1L downstream of RhoA.

Figure 3

a, Active RhoA and active PKD1 mediate SSH1L phosphorylation at S978. HeLa cells were co-transfected with myc-tagged SSH1L and vector, constitutively-active RhoA (RhoA.CA) or PKD1 (PKD1.CA). SSH1L was immunoprecipitated (α-myc) and samples were analyzed with α-pS978-SSH1L. Blots were re-probed for SSH1L (α-SSH1L). Control blots showing PKD1 and RhoA.CA transgene expression were performed by Western blotting using anti-PKD1 or anti-GST antibodies as indicated. b, Phosphorylation of endogenous SSH1L is dependent on RhoA. HeLa cells were transfected with vector control or active RhoA.CA and treated with C3 transferase as indicated. SSH1L was immunoprecipitated (α-SSH1L) and analyzed for phosphorylation at S978 (α-pS978). Blots were re-probed for SSH1L. Control blots showing RhoA.CA transgene expression were performed by Western blotting using α-GST antibodies. c, HeLa cells were transfected with RNAi control or PKD-RNAi. The next day cells were transfected with vector or constitutively-active RhoA (RhoA.CA). Endogenous SSH1L was immunoprecipitated (α-SSH1L) and analyzed for PKD1-mediated phosphorylation (α-pS978). Blots were re-probed for SSH1L (α-SSH1L). Control blots showing PKD1 (numbers show knockdown of PKD1 relative to control) and RhoA.CA transgene expression were performed by Western blotting using α-PKD1 or α-GST antibodies as indicated. A quantification of SSH1L phosphorylation at S978 from four different experiments is depicted in Fig. S6 (supplemental information). d, HeLa cells were co-transfected with vector control or PKD1.KD and constitutively-active RhoA (RhoA.CA). Endogenous SSH1L was immunoprecipitated (α-SSH1L) and analyzed for PKD1-mediated phosphorylation (α-pS978). Blots were re-probed for SSH1L (α-SSH1L). Control blots showing PKD1 and RhoA.CA transgene expression were performed by Western blotting using α-PKD1 or α-GST antibodies as indicated. Uncropped images of Fig. 3d are shown in Supplementary information Fig. S6.