Figure 2. Subcellular distribution of Rnd1 protein in rat brain. A, Specificity of an anti-Rnd1 antibody. COS-7 cells were transiently transfected with expression vectors encoding HA-tagged Rnd1, Rnd2, Rnd3, or an empty vector. Cell lysates were immunoblotted with antibody against Rnd1 (top) or HA epitope (bottom). B, Each subcellular fraction of P12 rat brain (80 μg of total protein) immunoblotted with the anti-Rnd1 antibody (top) or anti-PSD-95 antibody (bottom). S100, Cytosol; P100, crude membrane; P2, crude synaptosome; LP1, synaptosomal membrane; S1, homogenate without nuclei and large debris; S2, supernatant above P2 fraction. C, Endogenous Rnd1 protein in hippocampal cell cultures. COS-7 cell lysate expressing HA-Rnd1 and P100 fractions (45 μg of total protein) of dissociated hippocampal cell cultures at 17 and 31 DIV were immunoblotted with the anti-Rnd1 antibody. D, Immunofluorescence microscopy of dendrites of cultured hippocampal neurons. Cells at 17 DIV were fixed with methanol and immunostained. a, Cells were transfected with EGFP-F vector at 6 DIV. EGFP-F and Rnd1 labeling are shown in the top and middle panels, respectively. Bottom panels (Merge) show the superposition of the top and middle images. Rnd1 immunostains are observed in the dendritic shaft regions (arrows) including spine-like structures (arrowheads). b, PSD-95 and Rnd1 labeling were shown in the top and middle panels, respectively. Bottom panels (Merge) show the superposition of the top and middle images. Arrowheads or arrows indicate the regions in which Rnd1 immunostains are localized with or without PSD-95-immunopositive puncta, respectively. Scale bar, 5 μm.