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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

a, HeLa cells were transfected with vector, constitutively-active PKD1 (PKD1.CA) or kinase-dead PKD1 (PKD1.KD) and FLAG-tagged cofilin. Cofilin was immunoprecipiated (α-FLAG), cofilin phosphorylation was detected with α-pS3-cofilin antibody and samples were re-probed for total cofilin (α-FLAG). The expression of PKD1 was verified by Western blotting (α-PKD1). b, HeLa cells were transfected with control RNAi or PKD-RNAi. The next day, cells were transfected with FLAG-tagged cofilin. Cofilin was immunoprecipiated (α-FLAG), cofilin phosphorylation was detected with α-pS3-cofilin antibody and samples were re-probed for total cofilin (α-FLAG). The knockdown of PKD1 was verified by Western blotting (α-PKD1) and numbers show knockdown of PKD1 relative to control. A quantification of cofilin phosphorylation at S3 from three different experiments is depicted in Fig. S9A (supplemental information). c, HeLa cells were transfected with control RNAi or PKD-RNAi. The next day, cells were transfected with FLAG-tagged cofilin and constitutively-active RhoA (RhoA.CA). Cofilin was immunoprecipiated (α-FLAG), cofilin phosphorylation was detected by probing with α-pS3-cofilin antibody and samples were re-probed for total cofilin (α-FLAG). The expression of RhoA and the knockdown of PKD1 were verified by Western blotting (α-GST and α-PKD1). Numbers show knockdown of PKD1 relative to control. A quantification of cofilin phosphorylation at S3 from three different experiments is depicted in Fig. S9B (supplemental information). d, HeLa cells were co-transfected with FLAG-tagged cofilin, vector control, SSH1L, SSH1L.S978A and constitutively-active PKD1 (PKD1.CA) as indicated. Cofilin was immunoprecipiated (α-FLAG), cofilin phosphorylation was detected by probing with α-pS3-cofilin antibody and samples were re-probed for total cofilin (α-FLAG). The expression of PKD1 and SSH1L was verified by Western blotting (α-PKD1 or α-YFP). e, HeLa cells were transfected with vector control, constitutively-active PKD1 (PKD1.CA), RNAi control or PKD1-RNAi as indicated. Cell lysates were subjected to IEF-PAGE to separate endogenous S3-phosphorylated and unphosphorylated cofilin. Samples were analyzed with α-S3-cofilin and α-cofilin antibodies. The Western blot show representative experiments. The ratio of phospho-cofilin/cofilin of three independent experiments is depicted in the right panel. Uncropped images of Fig. 6a are shown in Supplementary information Fig. S6.

a, HeLa cells were transfected with vector, constitutively-active PKD1 (PKD1.CA) or kinase-dead PKD1 (PKD1.KD) and FLAG-tagged cofilin. Cofilin was immunoprecipiated (α-FLAG), cofilin phosphorylation was detected with α-pS3-cofilin antibody and samples were re-probed for total cofilin (α-FLAG). The expression of PKD1 was verified by Western blotting (α-PKD1). b, HeLa cells were transfected with control RNAi or PKD-RNAi. The next day, cells were transfected with FLAG-tagged cofilin. Cofilin was immunoprecipiated (α-FLAG), cofilin phosphorylation was detected with α-pS3-cofilin antibody and samples were re-probed for total cofilin (α-FLAG). The knockdown of PKD1 was verified by Western blotting (α-PKD1) and numbers show knockdown of PKD1 relative to control. A quantification of cofilin phosphorylation at S3 from three different experiments is depicted in Fig. S9A (supplemental information). c, HeLa cells were transfected with control RNAi or PKD-RNAi. The next day, cells were transfected with FLAG-tagged cofilin and constitutively-active RhoA (RhoA.CA). Cofilin was immunoprecipiated (α-FLAG), cofilin phosphorylation was detected by probing with α-pS3-cofilin antibody and samples were re-probed for total cofilin (α-FLAG). The expression of RhoA and the knockdown of PKD1 were verified by Western blotting (α-GST and α-PKD1). Numbers show knockdown of PKD1 relative to control. A quantification of cofilin phosphorylation at S3 from three different experiments is depicted in Fig. S9B (supplemental information). d, HeLa cells were co-transfected with FLAG-tagged cofilin, vector control, SSH1L, SSH1L.S978A and constitutively-active PKD1 (PKD1.CA) as indicated. Cofilin was immunoprecipiated (α-FLAG), cofilin phosphorylation was detected by probing with α-pS3-cofilin antibody and samples were re-probed for total cofilin (α-FLAG). The expression of PKD1 and SSH1L was verified by Western blotting (α-PKD1 or α-YFP). e, HeLa cells were transfected with vector control, constitutively-active PKD1 (PKD1.CA), RNAi control or PKD1-RNAi as indicated. Cell lysates were subjected to IEF-PAGE to separate endogenous S3-phosphorylated and unphosphorylated cofilin. Samples were analyzed with α-S3-cofilin and α-cofilin antibodies. The Western blot show representative experiments. The ratio of phospho-cofilin/cofilin of three independent experiments is depicted in the right panel. Uncropped images of Fig. 6a are shown in Supplementary information Fig. S6.