NSF knockdown results in decreased SERT expression at the plasma membrane in HEK293-hSERT cells. (A) Biotinylation experiments in HEK293-hSERT cells transfected with siRNA-2 targeting a specific NSF sequence or negative control. Transfected cells were incubated with sulfo-NHS-SS-biotin, and labeled proteins were analyzed by immunoblotting using anti-SERT antibodies. (B) Quantitation of relative band densities for SERT was performed by scanning densitometry. Data are expressed as the means ± standard deviation, n = 6 to 9. ***P < 0.001 vs negative control (two-tailed unpaired t-test). (C) Double immunocytochemical staining for SERT (green) and NSF (red) in HEK293-hSERT cells transfected with control siRNA (upper panels) and siRNA for NSF (siRNA-2, lower panels). Scale bar: 10 μm. Results are representative of three independent experiments. NSF, N-ethylmaleimide-sensitive factor; SERT, serotonin transporter; siRNA, small interfering RNA.