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Figure 4—figure supplement 2. Design and biochemical validation of concatemeric ASICs.(A) Cartoon illustrating three linked subunits, ‘a-c’ in a concatemeric channel. Lettering below shows nucleotide and protein sequence, containing: untranslated nucleotide sequence, restriction sites and peptide linkers (black); and ASIC1a sequence (blue). (B) Primer pairs used to generate fragments a-c for subsequent ligation into concatemeric constructs, including: nucleotide spacers and untranslated sequence (grey); restriction sites (orange); peptide linker (black); and ASIC1a sequence (blue). (C) Western blot, detecting ASIC1a amongst denatured total cell-surface protein from oocytes injected with indicated ASIC1a mRNAs. ‘WT’, regular ASIC1a; ‘WT-WT-WT’, concatenated wild-type subunits; ‘SA-WT-WT’, one of the three 1/3 S12’A concatemeric permutations tested (shown in Figure 4D), etc. The main band of protein from oocytes injected with regular ASIC1a mRNA is ~70 kDa, corresponding to single ASIC1a subunits. For protein from oocytes injected with concatemeric ASIC1a mRNA, the main band is ~210 kDa, corresponding to three covalently linked subunits in a concatemer. (D) Maximum current amplitude (‘I (μA)”) activated by pH 5.0 at oocytes injected with regular WT ASIC1a mRNA (‘WT’), concatemeric WT mRNA (‘WT-WT-WT’), concatemeric mutant mRNA, or nothing (‘uninjected’). Columns, mean current amplitude; circles, individual data points. Current amplitude at 3/3 L7’A-mutant and at 3/3 S12’A-mutant-injected oocytes was not discernable from uninjected oocyte.DOI:
http://dx.doi.org/10.7554/eLife.24630.010 Image published in: Lynagh T et al. (2017) © 2017, Lynagh et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View |