The Journal of Neuroscience, July 26, 2006, ():
Stargazin and Other Transmembrane AMPA Receptor Regulating Proteins Interact with Synaptic Scaffolding Protein MAGI-2 in Brain
J. Neurosci. Deng et al.
26: 7875
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplemental Figure 1. TARP mRNA expression is not altered by the stargazer mutation. A-D, horizontal serial sections (12 μm) from adult wildtype (+/+) and stargazer (stg/stg) brain were hybridized with radiolabeled antisense oligonucleotide probes for the mouse TARP genes Cacng2, -3, -4 and -8 and visualized by autoradiography. The stargazer Cacng2 mutation results in aberrant mRNA splicing and the absence of detectable γ2 protein without appreciably altering mRNA levels (Letts et al., 1998). Cacng2 mRNA was observed at highest levels in cerebellum but was also seen in most other brain regions examined, including cerebral cortex and hippocampus. Cacng3 was expressed at highest levels in cerebral cortex but also detected in olfactory bulb, hippocampus and striatum. Cacng4 is expressed at peak levels in the late embryo and decreases postnatally (Fukaya et al., 2005) where it retains low levels of expression in striatum, thalamus and olfactory bulb. Cacng8 is expressed at highest levels in hippocampus but is also observed in cerebral cortex, olfactory bulb, septum and striatum. We did not observe any obvious changes in the mRNA expression of the TARP genes that would preclude the use of adult stargazer mice as controls in other experiments. One of the two olfactory bulbs of the wild type brain was removed upon initial dissection to ensure later identification. The specificity of each probe was confirmed by the absence of hybridization in the presence of 100-fold excess unlabeled probe (data not shown). Supplemental methods: Antisense oligonucleotide DNA probes corresponded to nonhomologous portions of each mouse TARP transcript (Supplemental Table 1). Probes were end-labeled using terminal deoxynucleotidyl transferase (Promega, Madison, WI) and α-35S-dATP (1250 Ci/mmol; NEN) to a specific activity of 100 dpm/μg. Unincorporated nucleotides were removed using Bio-Spin 6 chromatography columns (Bio-Rad, Hercules, CA) and dithiothreitol (DTT) was added to a final concentration of 20 mM. Three month old stg/stg and wild type male littermates were sacrificed by cervical dislocation and the brains removed, immediately frozen on dry ice and embedded in Tissue-Tek O.C.T. Compound (Ted Pella, Redding, CA). Horizontal sections (12 μm) were cut using a cryostat microtome and thaw-mounted onto Superfrost/Plus glass slides (Fisher). Sections were fixed in 4% paraformaldehyde in PBS, rinsed in PBS and dehydrated in an ascending ethanol series. Hybridization and washes were performed as previously described (Burgess et al., 1999b). Sections were exposed to Kodak (Rochester, NY) BioMax MR film for 10 days. Autoradiographs were digitalized using SprintScan 35 (Polaroid, Cambridge, MA) and arranged using Photoshop (Adobe Systems, San Jose, CA). Supplemental reference: Burgess DL, Biddlecome GH, McDonough SI, Diaz ME, Zilinski CA, Bean BP, Campbell KP, Noebels JL (1999b) β subunit reshuffling modifies N- and P/Q-type Ca2+ channel subunit compositions in lethargic mouse brain. Mol Cell Neurosci 13:293-311.
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Supplemental Figure 2. Phylogram indicating similarity among the five MAGI-2 PDZ domains. The amino acid sequences of the five PDZ domains of mouse MAGI-2 (annotated within NCBI accession NM_015823.1) were aligned using the ClustalW v1.8 program (Thompson et al., 1994) and the Neighbor-Joining method was used to infer phylogenetic relationships among the domains as described in Burgess et al. (2001). MAGI-2 (M2) PDZ domains 1, 3, and 5 are most closely related to each other while PDZ domains 2 and 4 are more distantly related. The bar at bottom indicates 0.1 amino acid substitutions per position utilizing the PAM substitution matrix.
- supplemental material
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Supplemental Table 1. Oligonucleotide primers used in this study (submitted as a separate Microsoft Excel file).
