RIC-3 increases whole cell Fl-Bgt labelling and surface trafficking of α7 receptors but does not alter protein levels. Increasing concentrations of RIC-3 progressively augmented total Fl-Bgt binding under cell permeabilizing conditions (A, B). Fl-Bgt binding of surface α7 receptors also increased with RIC-3 but peaked at 1:1 RIC-3 to α7 and diminished at a 5:1 ratio (A, B). Total (A) and fold-change (B) of Fl-Bgt binding is shown. The surface and whole cell number of Fl-Bgt binding sites was quantified by measuring the integrated density of fluorescence intensities of Alexa 648-tagged α-bungarotoxin around the outer surface of each cell or within the cell, for non-permeabilizing and permeabilizing conditions, respectively. Significant difference levels comparing groups of RIC-3 coexpressing cells relative to no RIC-3 controls: * p < 0.05, ** p < 0.01, *** p < 0.001 Wilcoxon rank sum test post-hoc pair wise analysis. The number of cells analyzed for total receptor labelling in (A, B) are 0:1 (negative control, n = 14), 0.02:1 (n = 13), 0.1:1 (n = 14), 1:1 (n = 18), and 5:1 (n = 15). The number of cells analyzed for surface receptor labelling in (A, B) are 0:1 (n = 6), 0.02:1 (n = 8), 0.1:1 (n = 8), 1:1 (n = 11), and 5:1 (n = 11). (C, D) Mean emission intensity of the α7-Venus and α7-Cerulean fluorophores per HEK293T cell were determined at various concentrations of RIC-3. There was no significant change in either α7-Venus or α7-Cerulean fluorescent protein levels with various amounts of RIC-3 co-expressed inside the cells (p = 0.6, one-way ANOVAs; and p = 0.2, Kruskal-Wallis rank sum test, respectively).
Dau et al. BMC Neuroscience 2013 14:47 doi:10.1186/1471-2202-14-47