Cellular and subcellular localization of the HVRP1 protein.

A) Immunohistochemical analysis of HVRP1 distribution in cerebellar tissue. Cerebellar cortical section from a wild-type rat stained with polyclonal antibody raised against HVRP1 (red). GC: granule cell layer, PC: Purkinje cell layer, ML: molecular layer, scale bar: 50 µm. B) Higher magnification of granule neurons expressing HVRP1, from (A). Scale bar is 10 µm. C) Ultrastructural localization of HVRP1 in the rat cerebellar GC layer visualized with the pre-embedding immunogold method. Positive labeling is in black, in the glomeruli, at the dendritic claws of granule cells surrounding mossy fiber terminals. Scale bar is 0.1 µm. D) Basic circuit diagram of the cerebellar cortex showing the location of HVRP1 in the dendrites and cell bodies of GCs. GO: Golgi cell, GR: glomerulus, MF: mossy fiber, PC: Purkinje cell. E) Confocal images of cultured mouse cerebellar granule neurons fixed and immunostained at 8 DIV. HVRP1 signal is shown in red (antibody dilution 1∶1000), DAPI signal is in blue. F) Staining as in (E) but with anti-HVRP1 antibody at dilution 1∶500 pre-incubated with HVRP1 antigen peptide (see Methods). Scale bars are 10 µm. G) Confocal image of live HEK293A cells expressing recombinant EGFP-tagged human HVRP1 (green) and labeled with the plasma membrane marker FM-464 (red). Scale bar is 10 µm. H) Western blot of total protein extracts from HEK293A cells transfected with hHVRP1 (H) and non transfected (N). AP indicates pre-incubation of anti-HVRP1 antibody with antigen peptide.