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Fig. 4. GSK-3ß is membrane bound and accumulates in the Golgi region. (A) Hippocampal neurons were fixed and the localisation of endogenous GSK-3ß was detected by immunofluorescence. The Golgi was labelled by WGA-FITC. Single confocal sections are shown. (B) Neurons were solubilised for 5 minutes on ice in 0.1% Triton X-100, fixed in 4% PFA and GSK-3ß was visualised by immunofluorescence. (C) Cytosolic and membrane fractions were prepared by ultra-centrifugation at 100,000 g and analysed by western blot comparing equal amounts of total protein. N-cadherin and p38 MAP kinase were used as markers for membrane and cytosolic fractions, respectively. (D) The development of individual neurons (untreated control or treated from 24-48 hours with SB216763) was followed on cellocate (Eppendorf) coverslips. (E) Traffic of vesicles and membrane compartments in all neurites was imaged by phase-contrast microscopy in control and SB216763-treated hippocampal neurons. The number of membrane carriers travelling within 1 minute through a defined proximal and distal neurite segment (see Materials and Methods) were counted for all neurites. Numbers were sorted according the amount of membrane carriers counted. In control neurons, the first column represents the traffic in the axon, which was always the neurite with the most intense traffic (mean ± s.e.m., **P<0.001). Images of the experiment are shown in supplementary material, Fig. S4 and Movies 1-4. (F) Frequency distribution of the velocities of the membrane carriers measured in Fig. 4E. Fifty membrane carriers were tracked for each condition and the speed of anterograde or retrograde transport was measured. The overlapping peaks in the frequency-distribution diagrams show that there is no significant change in the distribution of velocities measured in control neurons and neurons treated for 48 hours with the GSK-3 inhibitor. (G) The growing axon was ablated and re-growth followed over 24 hours. The axon was marked by anti-Tau-1 antibody. Bars, 10 µm.
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