Figure 1.

Localization of Citron-N to dendritic spines and spine-associated Golgi. (A) Cultured hippocampal neurons (14 DIV) were immunostained with a Citron antibody (Cit-N) and Texas red isothiocyanate-conjugated phalloidin (PHD). Cit-N is localized in discrete clusters that are distributed along the dendritic shaft. A few of them colocalized with actin-rich protrusions (arrows). (B) In 21 DIV neurons processed as in (A), most of the Cit-N-reactive clusters colocalized with peripheral actin-rich structures (arrows). Scale bars, 10 μm. (C) High-magnification images of a 21 DIV primary hippocampal neuron dendrite co-stained for Cit-N (green), F-actin (blue) and TGN38 (red). The arrows indicate some of the structures in which all the analysed proteins colocalize. (D) A 50 μg portion of different fractions obtained from the adult mouse brain were analysed for levels of the indicated proteins by western blotting. Rib-II, ribophorin-II; T, total homogenate; P, low-speed pellet; S, low-speed supernatant; NF, nuclear pellet; Syn, crude synaptosomes; Cyt, cytosol. (E) A 1 mg portion of the crude synaptosomal fraction was immunoprecipitated (IP) with Cit-N antibodies or preimmune serum (Pre), and co-precipitation of the indicated proteins was analysed by western blotting. Data are representative of four independent experiments. Cit-N, Citron-N; DIV, days in vitro.