Anti-HA epitope antibody binding confirms no change in nAChR protein with 30 min nicotine and no change in nAChR conformation within 3 min of nicotine. (A) Equimolar amounts of α4CFP and β2YFP cDNA were transfected in HEK293T cells and time lapse imaging of FRET was performed at 30°C. Cells were imaged for FRET at 1 and 3 min before and 1 and 3 min during 10 μM nicotine incubation. There was no significant change (p = 0.88, one-way repeated measures ANOVA, n = 12) in FRET efficiency between nicotinic receptor subunits with bath applied nicotine during the 3 min nicotine incubation. (B) 30 min application of 10 μM nicotine did not alter anti-HA epitope antibody binding of HA epitope tagged α4 (α4-HA) and β2 (β2-HA) subunits expressed in HEK293T cells. (C) RIC-3 coexpression with α4-HA and β2-HA subunits resulted in significantly enhanced levels of anti-HA antibody labelling of α4-HA (p < 0.0001, Wilcoxon rank sum test) and β2-HA (p < 0.0001, Wilcoxon rank sum test) subunits as compared to cells not coexpressing RIC-3.
Dau et al. BMC Neuroscience 2013 14:47 doi:10.1186/1471-2202-14-47