NSF interacts with SERT in vivo . (A) Interaction of SERT with NSF in mouse brain. Immunoblot of total proteins from non-tcTPC- and tcTPC-treated mouse brains (as input, lanes 1 and 2, respectively). Proteins from non-tcTPC- or tcTPC-treated mouse brains were immunoprecipitated with SERT antibodies (lane 3 and 4), and the resulting immunoblot was probed for NSF. In immunoprecipitated samples using tcTPC-treated mouse brains, SERT–NSF complexes and free NSF were identified (lane 4). Results are representative of three independent experiments. (B) NSF co-localizes with SERT in primary cultures of mouse raphe nuclei neurons. Triple immunocytochemical staining for SERT (green), NSF (red) and 5-HT (blue) in primary cultures of mouse raphe nuclei neurons. The third panel (merged) shows that NSF co-localizes with SERT primary cultures of mouse raphe nuclei neurons. These neurons are 5-HT-positive serotonergic neurons (as shown in the fourth panel). Scale bars: 10 μm. Results are representative of three independent experiments. 5-HT, 5-hydroxytryptamine; IB, immunoblotting; IP, immunoprecipitation; MW, molecular weight; NSF, N-ethylmaleimide-sensitive factor; SERT, serotonin transporter; tcTPC, time-controlled transcardiac perfusion cross-linking.