Blocking Golgi–ER cycling prevents up-regulation of nAChRs. (A) Representative TIRFM images of α4-SEPβ2 (right) or α6-SEPβ2β3 (left) nAChR subunits. Nicotine (50 nM α6-SEPβ2β3 nAChRs or 100 nM α4-SEPβ2 nAChRs) and/or 20 µM CI-796 were added 24 h before imaging. Bars, 10 µm. Quantification of PMID for α6-SEPβ2β3 nAChRs (B₁) or α4-SEPβ2 nAChRs (B₂) in Neuro-2a cells with drug treatments: no drug, nicotine, CI-976, or combined (n = 17–23). *, P < 0.05; ***, P < 0.0001 (ANOVA).