Mouse neuroblastoma 2a (Neuro-2a) cells were cultured using standard techniques (Xiao et al., 2011 (link)). Cells were plated by adding 90,000 cells to poly-d-lysine–coated 35-mm glass-bottom imaging dishes (MatTek Corporation) and cultured in a humidified incubator (37°C, 95% air, 5% CO2). Cells were transfected with 500 ng of each nAChR subunit plasmid and 250 ng GalT-mCherry or Sec24D-mCherry for assays. Plasmids were mixed with 250 µl Opti-MEM. Lipofectamine-2000 was separately added to 250 µl Opti-MEM. After 5 min at 24°C, DNA and Lipofectamine solutions were mixed together and incubated for 25 min at 24°C. The solutions were then added to preplated Neuro-2a cells and incubated for 24 h. After 24 h, the Opti-MEM was removed and replaced with growth medium. 50 or 500 nM of filter-sterilized nicotine was added after replacing the Opti-MEM with standard culture medium (α6β2β3 nAChRs). For α4β2 nAChRs, 100 nM nicotine was used for 48 h (nicotine was added at the time of transfection and then replenished when the media was changed). 20 µM CI-976 was added with nicotine 24 h before imaging. Cells were imaged 48 h after transfection.