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Fig. 1. Developmental fluctuation of stathmin expression in Purkinje cells (PCs) and phosphorylation of stathmin at Ser16 in cerebellum. (A) Schematic representation of domain organization of stathmin. N-terminal regulatory domain contains four serine residues as phosphorylation sites. The C-terminal interaction domain has two helical structures and interacts with two 
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-tubulin heterodimers. (B) PC collection from P12 or P15 frozen cerebellar sections (30 µm thick, stained with Toluidine Blue) using the Laser Capture Microdissection System, LM2000. (–) PC (left photo) shows a section after laser treatment, with the PCs removed. PC (right photo) shows captured PCs. (C) PC-specific differential display (PC-DD) showing downregulation of stathmin expression between P12 and P15. Total cellular RNAs were prepared independently from the PCs of four animals (two at P12 and two at P15) and subjected to PCR-differential display. Arrowhead indicates stathmin cDNA, corresponding to nucleotides 782 to 963 of mouse stathmin 1 (GenBank accession number: NM019641). (D) In situ hybridization was carried out with a stathmin antisense probe and frozen cerebellar sections (10-µm thick). Brains were dissected at P12, P15 and P18. Lobule 4-5 (L4-5) is shown. (E) The average signal intensity of stathmin mRNA in PC somata of L4-5 was quantified. Data from each time point were obtained from 114-128 cells from two animals. Data are shown as mean ± s.e.m. P values of Student's t-test are given in the graph. (F) Immunohistochemical analysis of stathmin level in cerebellar cortex at P12 and P18. Micrographs of brain sections from P12 and P18 mice mounted on the same slide were photographed on a laser-scanning confocal microscope under the same conditions. Rightmost panels are higher-magnification images of merged micrographs. Thus, the intensities of the stathmin signals from P12 and P18 can be compared quantitatively. ML, molecular layer; PCL, PC layer; EGL, external granule cell layer; IGL, internal granule cell layer. Bars, 50 µm (D,F). (G,I) Immunoblotting of whole cell lysate prepared from P12, P15 and P18 cerebellum. Anti-stathmin antibody and anti-phospho-stathmin (Ser16) antibody were used in G and I, respectively. An almost equal amount protein was loaded in each lane. Alpha-tubulin provided a loading control. (H,J) Signal level normalized to -tubulin level was quantified from three independent samples of different animals at each time point. Error bars indicate mean ± s.e.m.
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