The Journal of Neuroscience, June 13, 2007, ():
Cytoplasmic Polyadenylation Element Binding Protein 1-Mediated mRNA Translation in Purkinje Neurons Is Required for Cerebellar Long-Term Depression and Motor Coordination
J. Neurosci. McEvoy et al.
27: 6400
Supplemental Data
Files in this Data Supplement:
- supplemental material
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Supplemental Figure 1. Construction and Expression of the L7-GFP-mCPEB1-AA Transgene. A schematic of the L7 gene shows the exon sequence, start codon and BamHI insertion site. The L7?AUG vector is shown in the bottom panel where all potential start codons (AUG) in all reading frames were eliminated by PCR. As a result, translation is only initiated from a start codon provided by the inserted cDNA sequence. The L7-GFP-mCPEB-AA transgene was inserted into a unique BamHI site in the L7?AUG vector following BglII digestion of the transgene.
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Supplemental Figure 2. Protein synthesis inhibition and Aurora kinase inhibition have similar affects on cerebellar LTD. (A) Protein synthesis inhibition immediately following stimulation results in a cerebellar LTD deficit similar to protein synthesis inhibition during the time of stimulation. Since the time course of the inhibition of LTD in our experiments was significantly faster than that seen in in vitro experiments and there was a possibility that cycloheximide might have unintended affects on PNs (Linden, 1996), we applied the mRNA translation inhibitor anisomycin (50?m) to the bath for the 10 mins. immediately following conjunctive stimulation. The initial amplitude of the induced depression was greater than that seen when cycloheximide was in the bath prior to stimulation, but the time course of EPSC amplitude returning to baseline levels was similar (n=7, mean +/- SEM). Indicating that rapid protein synthesis is necessary to maintain the initial depression, consistent with results reported by the Ito group (Karachot et al., 2001). (B) ZM447439 is a quinazoline derivative that blocks Aurora kinases with an IC50 of 110nM. Therefore, to confirm that hesperadin inhibition of LTD (Figure 4) was likely the result of its affect on Aurora kinase we again induced LTD in the presence of ZM447439 (100nM) in the recording patch pipette. LTD is inhibited with a similar time course to that of hesperadin (n=5 cells from 3 mice; data is mean + SE.
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Supplemental Figure 3. Control staining for the Aurora A immunohistochemistry. Aurora A staining was performed as described in Figure 4. Sections were treated with IgG (control) at the same concentration as the anti-Aurora A antibody followed by species-specific secondary conjugated to FITC. The images were taken at the same exposure time and processed identically in Photoshop. Cerebellar molecular layer (ML) and granule cell layer (GCL) are identified by vertical lines, and the Purkinje cell layer (PC) is also indicated. Scale bar=50?m.
