Figure 6

Kal7 and Rac1 are enriched in mouse striatal PSDs. Adult mouse striatum homogenized in buffer containing 0.32 M sucrose was subjected to differential centrifugation as described in Methods. After hypotonic lysis, synaptosomal membranes were pelleted, resuspended and subjected to equilibrium sucrose density centrifugation. Fractions were collected from the top (Sample) to bottom [Pellet (mitos)] of the gradient; the fraction taken from the interface of the 1.0/1.2 M sucrose layers was extracted with TX-100, yielding a TX-soluble fraction (TxS) and the PSD fraction. Equal amounts of protein (5 μg) from each fraction were subjected to SDS-PAGE; Kalirin was visualized using the Kal-spectrin antibody and the Kal7 antibody. The NR2B subunit of the NMDA receptor served as a PSD marker; BiP served as an endoplasmic reticulum marker. Rac1, one of the substrates of Kal-GEF1, was also localized to PSDs. Cyt = cytosol; ER/G = endoplasmic reticulum/Golgi-enriched fraction.