Figure 2.

Chmp2b forms clusters in dendrites. A, Cultured mouse cortical neurons were lysed at various stages of maturation, and equal amounts of lysate protein were analyzed by immunoblotting with the indicated antibodies. PSD-95 was used as a marker for synaptogenesis. B, Hippocampal neurons were transfected with empty pSuper-mCherry vector, fixed at 21 DIV, and stained by dual immunofluorescence with anti-Chmp2b and anti-PSD-95 antibodies. Left, Punctate staining of a dendritic segment in a transfected neuron (single confocal plane). Staining of nontransfected cells is also visible. Merge, Overlap (yellow) between Chmp2b and PSD-95 puncta, in dendritic shafts and spines, visualized with mCherry (white contour). The numbered arrowheads indicate spines with colocalized puncta. Right, Enlarged views of the same spines. Scale bar, 20 μm. C, Top, Dendritic segment double stained by anti-Chmp2b and anti-PSD-95. Bottom, Same after randomization of Chmp2b staining pattern. The correlation coefficient (r) between actual Chmp2b and PSD-95 staining intensities is significantly higher than the mean r value generated by randomized Chmp2b patterns (p < 0.0001, z test). D, Specificity of Chmp2b immunofluorescence. Hippocampal neurons were transfected with pSuper-mCherry expressing Chmp2b-specific shRNA, stained by immunofluorescence with anti-Chmp2b, and imaged by confocal microscopy. Left, Representative transfected neuron, visualized by mCherry. Middle, Chmp2b immunostaining of the same field. Arrowheads indicate the position of the transfected cell dendrites and soma. N, Neighboring, nontransfected neuron. Right, Immunostaining (red hot pseudocolor) is mostly absent from the transfected neuron (outlined in white), though clearly detectable in the nontransfected cell (N). Scale bar, 10 μm.