The Journal of Neuroscience, November 9, 2005, ():
The Role of Snapin in Neurosecretion: Snapin Knock-Out Mice Exhibit Impaired Calcium-Dependent Exocytosis of Large Dense-Core Vesicles in Chromaffin Cells
J. Neurosci. Tian et al.
25: 10546
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplemental Figure 1.
(A) Electron micrographs of immature synapses from E18.5 hippocampus of snapin homozygous null mutants (upper) and wild-type (lower). The whole heads of E18.5 fetuses were fixed in 4% glutaraldehyde in 0.1 N sodium cacodylate buffer at pH7.4 for 1 hr at room temperature and then stored in fixative buffer at 4°C. Tissue blocks containing hippocampus were dissected, and coronal 60 mm sections were cut with a Vibratome and collected in 0.1 M PBS and then processed for EM as described in Methods.
(B) Snapin is expressed in chromaffin cells. A single Snapin band (223 bp) was amplified with RT-PCR from a single mouse chromaffin cell (lane 1), 10 ng total RNA of adrenal gland (lane 3) and cerebellum (lane 5) after the second PCR reaction, and amplified PCR product of Snapin-pCRII plasmid as control (lane 7). Lanes 2, 4, 6, and 8 are the PCR products digested with Taq I, which specifically cuts the 223-bp Snapin PCR products from lane 1, 3, 5, and 7 into two pieces (104 and 119 bp). M, molecular weight marker PUC19 DNA/MspI.
- supplemental material
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Supplemental Figure 2. GST pull-down analysis of the interaction between Snapin and SNAP-25. GST fusion proteins (~3 μg) and GST control were immobilized on Glutathione-Sepharose beads and then incubated with adult mouse brain homogenates. Bound protein complexes were immunoblotted with the antibodies against SNAP-25 and synaptotagmin-1 as indicated. B, bound protein; S, supernatant remaining after pull-down and loaded as 2 % with respect to the total homogenates used in each pull-down. In all experiments, a Triton X-100 extract of mouse brain homogenate was used as the source for natives SNAP-25 and synaptotagmin.
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