SUMOylated CASK proteins are present in the mouse brain. (A) Mouse brain homogenates purified from three different mice in the presence or absence of N-ethylmaleimide were immunoprecipitated with CASK antibody. The precipitates were then analyzed by immunoblotting using SUMO1 and CASK antibodies sequentially. The exposure time for each image is indicated. (B) Mouse brain subcellular fractions prepared as described in Materials and methods were used to examine the neuronal distribution of SUMOylated CASK. Equal amounts of each fraction were analyzed by immunoprecipitation-immunoblotting analysis as described in A. H, total homogenate; P1, nuclei and cell debris; S1, supernatant of P1; P2′, washed crude synaptosomal fraction; S2, supernatant of P2′; LP1, lysed synaptosomal membrane; LS1, supernatant of LP1; P3, light membrane fraction; S3, soluble cytosol. The asterisk indicates an unknown protein species recognized by SUMO1 antibody. (C) The percentage of SUMOylated CASK in total CASK proteins of each subcellular fraction. The protein levels of SUMOylated CASK and nonSUMOylated CASK were analyzed using ImageJ. For each fraction, the percentage of SUMOylated CASK was determined by dividing the amounts of SUMOylated CASK by the sum of SUMOylated and nonSUMOylated CASK. The data presented are the mean ± SEM (error bars) of four independent experiments. (D) SUMOylated CASK is mostly present in synaptic cytosol, LS2 fraction. The LS1 fraction was further separated into LP2 fraction, crude synaptic vesicles, and LS2 fraction, synaptic cytosol. CASK proteins were immunoprecipitated from equal protein amounts of LP1, LP2, and LS2 fractions and analyzed by immunoblotting using SUMO1 and CASK antibodies as indicated. The percentage of SUMOylated CASK in total CASK proteins is also indicated. (A, B, and D) Arrows indicate the positions of SUMOylated CASK; arrowheads point to the positions of unmodified full-length CASK.