Measuring hERG Endocytic Recycling

hERG endocytic recycling was measured using a modified sandwich cell-surface ELISA assay described previously^{37 (link)}. Briefly, cell-surface hERG was labelled with anti-HA primary antibody (1 h, on ice). Ab-hERG complexes were then internalized for 20 minutes at 37 °C; complexes remaining on the cell surface were then blocked with mouse monovalent F(ab′)₂ fragments (1:100; Jackson ImmunoResearch Laboratories, 1 h on ice). Recycling of the internalized Ab-hERG complexes was enabled by incubating cells at 37 °C for 0–20 minutes. Exocytosed Ab-hERG complexes were detected with HRP-conjugated secondary F’(ab)₂ as described above. Background signal was measured using a non-specific primary Ab as described above. Blocking efficiency with mouse monovalent F(ab′)₂ fragment was determined to be over 95% (data not shown). The size of the endocytic hERG pool following 20-minute incubation at 37 °C was measured in parallel, with recycling efficiency being expressed as percent of this pool.