Ewald RC , Cline HT .

R01 EY011261 NEI NIH HHS

	Figure 1. Detection of NMDAR subunits and splice variants in X. laevis tadpole brain. (A) Immunoblots for NR1, NR2A and NR2B were done on whole brain lysates of stage 47/48 tadpoles (Xen) with antibodies generated against homologous rat proteins. Immunoreactive bands of about the same size as in rat whole brain lysate (Rat) were found for all three subunits. (B) Western blots of whole cell lysates of HEK293 cells transfected with X. laevis the NR1-4a/b splice variant or NR2A cDNA, and their mock-transfected controls. (C) Cartoon of NR1 splice variants and the alternatively spliced exons (N1, C1, C2) and C-terminal ends (C2′) that they contain. (D) Cross-reactive bands for exons N1 and C1, as well as alternative C-terminal end C2′ in X. laevis were detected with antibodies to rat homologs by Western blot on whole brain extracts from stage 47/48 tadpoles and rat. No cross-reactive band was detected for C2. (E,F) C-terminal ends of NR1 mRNAs were amplified by RT-PCR and PCR with primer pairs 5′ to the spliced region and in the 3′UTR. (E) Twenty-six out of 29 nucleotide sequences contained only C2′ (Xen_NR1_C2′). They aligned perfectly to the published X. laevis NR1 sequence (Xen_NR1_X94156). (F) Two out of 29 nucleotide sequences contained C1 and C2′ (Xen_NR1_C1_C2′), indicating that NR1-3a/b also exists in X. laevis. The alignment to rat NR1-3a (Rat_NR1-3a) shows a very high degree of sequence conservation. Identical residues are marked with (*).
	Figure 2. Phylogenetic trees of NR2A and NR2B. The phylogenetic relationships of 11 NR2A and 14 NR2B proteins from different species and the NR2 proteins from C. elegans and D. melanogaster were analyzed using the Neighbor Joining algorithm. The confidence values of the nodes were determined after bootstrapping with 1000 repetitions and are shown at the nodes. The trees were rooted by aligning two unrelated sequences, mouse GABA-A-a1 and GABA-A-a2 (gray italics). The cloned X. Laevis NR2A and NR2B are bold, sequences that were assembled by hand are in italics and the remaining sequences have GenBank accession numbers.
	Figure 3. Conserved residues in X. laevis NR2A and NR2B. (A) Cartoon of the annotated functional domains and residues of NR2A. (B) Table of the number and percentage of conserved residues and domains of NR2A from 11 different species. The overall amino acid identity was determined in comparison to the X. laevis NR2A protein. (C) Cartoon of the annotated functional domains and residues of NR2B. (D) Table of the number and percentage of conserved residues and domains of NR2B from 14 different species. The overall amino acid identity was determined in comparison to the X. laevis NR2B protein. The number or percentage of conserved residues was determined in comparison to the sequence of the species where they were first characterized (mostly rat). Functional sites that are not conserved between all species are highlighted in gray.
	Figure 4. Protein sequence alignment of NR2A from 11 different species. The protein sequences of X. laevis NR2A and 10 other species were aligned with the ClustalW algorithm. When full sequences were not available or could not be assembled by hand, incomplete sequences were used. Amino acids identical to the reference sequence are depicted as (.). Lack of sequence is symbolized by (−). A (*) below the alignment signifies amino acid identity, while (:) and (.) indicate the degree of amino acid similarity. NR2A is highly conserved between species. The membrane-associated segments M1–M4, other structurally and functionally important domains and amino acids, as well as known protein–protein interaction domains and phosphorylation sites are highlighted. Descriptions in italics indicate sites that are not conserved between species.
	Figure 5. Protein sequence alignment of NR2B from 14 different species. The protein sequences of X. laevis NR2B and 13 other species were aligned with the ClustalW algorithm. When full sequences were not available or could not be assembled by hand, incomplete sequences were used. Amino acids identical to the reference sequence are depicted as (.). Lack of sequence is symbolized by (−). A (*) below the alignment signifies amino acid identity, while (:) and (.) indicate the degree of amino acid similarity. NR2B is very highly conserved between species. The membrane-associated segments M1–M4, other structurally and functionally important domains and amino acids, as well as known protein–protein interaction domains and phosphorylation sites are highlighted. Descriptions in italics indicate sites that are not conserved between species.

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