Fig. 3.

LINGO1 suppresses cell surface expression of BKα channels. (A) Representative confocal sections from HEK293 cells expressing BK channels alone (Upper) or coexpressed with LINGO1 (Lower) in HEK293 cells. Cells were fixed and immunostained for LINGO1 and BKα channels and nuclear stain (TOPRO). (Scale bar, 7 μm.) (B) Representative experiment (Left) from an OCW assay to detect cell surface expression of BK channels in HEK293 cells in the presence of different concentrations of LINGO1 cDNA, run in quadruplicate. Surface expression (Flag-) was determined in nonpermeabilized cells probing for the extracellular Flag- epitope on the BKα channel N terminus. Total BKα expression was determined in the same well after cell permeabilization and probing for the -HA epitope on the intracellular C terminus of the BK channels. Quantification of BK channel surface expression, expressed as a fraction of the Flag/HA ratio in the absence of LINGO1 (Upper), and total BKα channel expression (Lower) expressed as a fraction of total BK channel in absence of LINGO1. Data are mean ± SEM from four to seven independent experiments in each group. *P < 0.05, **P < 0.01 vs. BKα alone ANOVA with post hoc Tukey test.