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13 Nutlin-3 Lies Unwrapped

Both approaches suggest that CELF4 nominally binds a vast number of mRNAs �C up to 15�C20% of the approximately 14,000 transcripts expressed. This includes Scn8a, which encodes Nav1.6 (Fig. 3A). Interestingly, the abundance of Scn8a RNA transcript was increased modestly in brain cDNA of Celf4 null compared with wild-type �C by about 20% (data not shown). Given that Nav1.6 is the primary determinant of AP initiation and main contributor of INaP in excitatory neurons, we thought there might be a more significant accumulation of Nav1.6 at neuronal sites critical for AP initiation and sought to determine this by immunostaining. The AIS is the preferred site RG7422 mw for the AP initiation in excitatory neurons and plays a critical role in excitability because this specialized axonal region contains a high density of Nav1.6 and other Na+ channel subtypes (Moore et al. 1983; Mainen et al. 1995; Rapp et al. 1996; Hu et al. 2009). We performed immunostaining of layer V pyramidal neurons of visual cortex to map the expression of Nav1.6 and Na+ channel-associated protein AnkG, which is also known to be enriched at the AIS (Fig. 4A). To measure the immunofluorescence intensity of Nav1.6 and AnkG quantitatively, Nutlin3 each well-defined AIS was traced, using AnkG as a mask, starting from the axon hillock and extending to the tapered AIS end (note that any AIS that was clearly truncated with a blunt end was excluded from further analysis), and the fluorescence intensity of each traced pixel was determined. Two image channels were simultaneously measured �C green for Nav1.6 and red for AnkG. We observed a very significant, Celf4 gene dosage-dependent increase in Nav1.6 expression at the AIS, using a relatively large sampling (n= 178 for +/+, 181 for +/? and 179 for ?/?). Importantly, this increase was also observed in Celf4 heterozygotes (Fig. 4A and B). The total fluorescence intensity for Nav1.6 was 52.6% higher in heterozygotes and 73.8% higher in null (P < 0.0001 for each, t test). The relative distribution Fluorouracil purchase of Nav1.6 along the AIS was unaltered in mutant neurons (Fig. 4C). Interestingly, AnkG protein was also more highly expressed in the AIS of Celf4 heterozygotes across the entire AIS, with a slightly larger increase in Celf4 heterozygotes (Fig. 4A, B and D). Ank3 (encoding AnkG), also a likely CELF4 target (Fig. 3B), is a scaffolding protein that recruits and concentrates sodium channels at the AIS. Thus, the apparently selective increase in AnkG at the AIS may contribute to or facilitate increased accumulation of Nav1.6 (Srinivasan et al. 1988; Zhou et al. 1998; Garrido et al. 2003; Lemaillet et al. 2003). AnkG also plays a role in suppressing INaP via its interactions with Nav1.6, which may explain why AnkG abundance is higher in heterozygotes compared with null, as it would be expected to respond to increased INaP (Shirahata et al. 2006).
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