Fig. 1. Sequence analysis and cloning of mouse Panx1 and Panx3. (A) Sequence alignment of mouse Panx1 and Panx3. ClustalW alignment and BLAST analysis of the two protein sequences showed 41% identity at the amino acid level (black boxes). Some regions of high homology coincide with the four transmembrane domains (TM1-TM4) predicted by the Toppred algorithm. Asterisks mark conserved cysteine residues present in both predicted extracellular loops. Underlined sequences in the carboxyl-tail indicate the peptides used for the generation of polyclonal antibodies for each pannexin. Gray boxes indicate conserved amino acid substitutions. (B) Based on sequence analysis, both Panx1 and Panx3 are predicted to be polytopic and contain several predicted N-glycosylation (red and orange residues) and phosphorylation (black) sites. Orange residues indicate extracellular N-glycosylation sites used for site-directed mutagenesis. (C) RT-PCR products generated with specific primers designed to amplify the entire coding regions of each pannexin. A 1.5 kb band corresponding to Panx1 was amplified from both brain and osteoblast RNA from 3-week-old mice. A 1.4 kb Panx3 product was amplified from osteoblast RNA. All bands were sequenced to verify their identity as pannexin transcripts, but only the clones containing the entire coding region were engineered into expression vectors for further analysis.