Figure 7.

nPIST and stargazin cofractionate and coimmunoprecipitate from brain. A, Stargazin and nPIST coimmunoprecipitate from the brain. Rat brain membranes were solubilized with SDS. After the addition of Triton X-100, extracts were immunoprecipitated with antibodies to either PIST (IP nPIST), preimmune sera (Pre), or stargazin-specific antibodies (IP stargazin). After SDS-PAGE, blots were probed with antibodies to PIST (nPIST), stargazin, PSD-95, GluR1, or synaptophysin. PIST antisera avidly precipitated nPIST and also coimmunoprecipitated stargazin, PSD-95, and GluR1 but not synaptophysin. Stargazin antiserum immunoprecipitated stargazin and also precipitated nPIST, PSD-95, and GluR1 but not synaptophysin. B, nPIST cofractionates with stargazin in brain membranes and coimmunoprecipitates GluR1 and PSD-95 from different fractions. Crude membranes were solubilized with Triton X-100 or SDS as described in Materials and Methods. nPIST and stargazin were present in both Triton X-100-soluble and SDS-soluble fractions, whereas PSD-95 was enriched in SDS-soluble and GluR1 was enriched in Triton X-100-soluble membranes. PIST antisera (IP nPIST) effectively coimmunoprecipitated stargazin from both fractions, PSD-95 from SDS soluble membranes, and GluR1 from Triton-soluble membranes. Synaptophysin was present in the Triton-soluble fraction but was not coimmunoprecipitated with PIST antiserum. For quantitation, see supplementary Table 1 (available at www.jneurosci.org/cgi/content/full/24/34/9491/DC1).