Figure 5. SUMOylation does not promote nuclear distribution of CASK but interferes with the interaction between CASK and protein 4.1N in COS cells. (A) Confocal analysis of the subcellular distribution of CASK proteins. Myc-tagged wild-type and CASK mutants were transfected into COS1 cells as indicated. Nuclei were counterstained with DAPI. (B) The interaction between protein 4.1N and CASK. COS1 cells were cotransfected with 0.25 μg FLAG-tagged protein 4.1N and either 1 μg Myc-tagged CASK or 1 μg CASK mutants as indicated. Immunoprecipitation was performed using FLAG tag antibody. The precipitates were analyzed by immunoblotting using specific antibodies to the Myc and FLAG tags sequentially. The asterisks indicate the residual Myc immunoreactivities from the top panel. The protein levels of coimmunoprecipitated CASK were analyzed by ImageJ version 1.38X (National Institutes of Health). The data presented are the mean ± SEM of three independent experiments. The relative amounts of proteins were normalized to the amounts of CASK input and precipitated protein 4.1 as indicated. The significance of any difference was determined by t test using SPSS software, and significant differences are shown; *, P < 0.05. (C) Overexpression of SUMO1 reduces the interaction between CASK and protein 4.1N. COS cells were triple transfected with CASK, flag-tagged protein 4.1N, and GFP-tagged SUMO1 or vector control and analyzed by immunoprecipitation-immunoblotting using antibodies as indicated. The DNA amounts for transfection are also shown. Because cotransfection with protein 4.1N reduces CASK amounts in the Triton X-100–solubilized lysate (see B), the DNA amounts for transfection were adjusted accordingly to make the CASK protein amounts similar among different groups. The relative protein amounts of CASK coimmunoprecipitated with protein 4.1N were normalized to both precipitated protein 4.1N amounts and CASK input amount. The data are means of two independent experiments. The asterisks indicate the nonspecific signals contributed by precipitated protein 4.1N. (D) SUMOylation reduces the association of CASK with actin cytoskeleton. COS cells were cotransfected with protein 4.1N and CASK constructs as indicated. Immunoprecipitation was performed using actin antibody and analyzed by immunoblotting using antibodies as indicated. The amount of CASK protein coimmunoprecipitated with actin, normalized to actin precipitated amounts, is shown. Data are the means of three independent experiments. Error bars indicate SEM. *, P < 0.05; **, P < 0.005; ***, P < 0.001. Bars, 20 μm.
Figure 5.

SUMOylation does not promote nuclear distribution of CASK but interferes with the interaction between CASK and protein 4.1N in COS cells. (A) Confocal analysis of the subcellular distribution of CASK proteins. Myc-tagged wild-type and CASK mutants were transfected into COS1 cells as indicated. Nuclei were counterstained with DAPI. (B) The interaction between protein 4.1N and CASK. COS1 cells were cotransfected with 0.25 μg FLAG-tagged protein 4.1N and either 1 μg Myc-tagged CASK or 1 μg CASK mutants as indicated. Immunoprecipitation was performed using FLAG tag antibody. The precipitates were analyzed by immunoblotting using specific antibodies to the Myc and FLAG tags sequentially. The asterisks indicate the residual Myc immunoreactivities from the top panel. The protein levels of coimmunoprecipitated CASK were analyzed by ImageJ version 1.38X (National Institutes of Health). The data presented are the mean ± SEM of three independent experiments. The relative amounts of proteins were normalized to the amounts of CASK input and precipitated protein 4.1 as indicated. The significance of any difference was determined by t test using SPSS software, and significant differences are shown; *, P < 0.05. (C) Overexpression of SUMO1 reduces the interaction between CASK and protein 4.1N. COS cells were triple transfected with CASK, flag-tagged protein 4.1N, and GFP-tagged SUMO1 or vector control and analyzed by immunoprecipitation-immunoblotting using antibodies as indicated. The DNA amounts for transfection are also shown. Because cotransfection with protein 4.1N reduces CASK amounts in the Triton X-100–solubilized lysate (see B), the DNA amounts for transfection were adjusted accordingly to make the CASK protein amounts similar among different groups. The relative protein amounts of CASK coimmunoprecipitated with protein 4.1N were normalized to both precipitated protein 4.1N amounts and CASK input amount. The data are means of two independent experiments. The asterisks indicate the nonspecific signals contributed by precipitated protein 4.1N. (D) SUMOylation reduces the association of CASK with actin cytoskeleton. COS cells were cotransfected with protein 4.1N and CASK constructs as indicated. Immunoprecipitation was performed using actin antibody and analyzed by immunoblotting using antibodies as indicated. The amount of CASK protein coimmunoprecipitated with actin, normalized to actin precipitated amounts, is shown. Data are the means of three independent experiments. Error bars indicate SEM. *, P < 0.05; **, P < 0.005; ***, P < 0.001. Bars, 20 μm.

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