Fig_3.tif (3.13 MB)

ASIC1 oligomeric states resolved by SDS-PAGE after crosslinking with BMOE.

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posted on 07.08.2015 by Miguel Xavier van Bemmelen, Delphine Huser, Ivan Gautschi, Laurent Schild

A: Anti-Anti-His-tag western blot of the surface-biotinylated protein fractions from either non-injected oocytes (n.i.), or oocytes expressing either the His8-tagged form of ASIC1a-ΔCCt (ΔCCt), lacking cysteines in the C-terminus, or the corresponding cysteine-substitution mutants V74C-ΔCCt, Y426C-ΔCCt, G430C-ΔCCt, and G433C-ΔCCt. Crosslinking with 2 mM BMOE was performed at the cell surface; the cell-surface biotinylated proteins were affinity-purified on streptavidin beads. Numbers I to IV, designate the four most prominent bands. B: Apparent Mw values of the ASIC1 oligomers (kDa, mean ±SD) estimated for each of the four main bands (I to IV) for the different constructs, as in A. Lines represents linear regression of the average values, with slopes ranging from 71 ± 4.2 to 73.8 ± 4.6 kDa for the different ASIC1a cDNA constructs.