Abstract

<p>(<b>A</b>) Western blot depicting fragmentation of α4-subunit seen as two bands in N-Flag-α4β3 and α4β3N-Flag-δ receptors reconstituted into CHAPS/asolectin micelles is presented. Both α4 bands are identified by polyclonal anti-α4 and monoclonal anti-Flag antibodies in the former receptor, confirming identity of the band. Numbers on the side indicate the position of the molecular weight markers (kDa). (<b>B</b>) Cells induced to express indicated GABA<sub>A</sub>Rs were prepared for Western blotting by either 1. suspending cells in suspension buffer; 2. suspending and sonicating; 3. leaving in a monolayer (untreated); or 4. lysing directly in the well with suspension buffer supplemented with 10 mM DDM; as indicated, prior to lysing cells with a 4x Laemmli sample buffer with 10% β-mercaptoethanol. Suspension buffer was supplemented with Protease Inhibitor Cocktail (Sigma) at 1:100 dilution. (<b>C</b>) Representative Western blot of samples obtained during α4β3N-Flag-δ receptor purification, as described in the materials and methods section. Numbers under each lane indicate the fraction the lower band comprises of the higher band, expressed as percentile points. (<b>D</b>) Membrane fraction from the α4β3N-Flag-δ was incubated for 1 hour at indicated temperatures prior to analysis by Western blotting. All blots are presented as grayscale and were uniformly adjusted for brightness and contrast to facilitate analysis. Full immunoblots used to make panels A-D are presented as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191583#pone.0191583.s004" target="_blank">S4</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191583#pone.0191583.s007" target="_blank">S7</a> Figs.</p

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