Co-immunoprecipitation (co-IP)

HEK293 were grown in 12-well culture plates and transfected using GeneCellin (Bulldog Bio) and 1.4 µg plasmid/well (pcDNA3-mDAT, pBABE-σ₁R, and pcDNA3-σ₁R-EYFP). Transformed cells were selected with 800 µg/ml Geneticin (G418) (Sigma Aldrich) 36–48 h later. Expression of stable cells was confirmed by Western blot. For co-IP experiments, stable cell lines were plated on 60 mm plates and transiently transfected 48 h prior and maintained under selection with 400 µg/ml Geneticin (G418). Co-IP experiments were carried out as outlined by the manufacturer (Co-Immunoprecipitation Kit; Thermo Scientific). Immunoprecipitation of equal amounts of mDAT, σ₁R, and σ₁R-EYFP expressing lysates was conducted for 14–18 h at 4 °C using AminoLink Plus Coupling Resin (ThermoFisher) in serum containing rabbit polyclonal antibodies against DAT (Poly16) raised in (kind gift from Dr. Roxanne Vaughan⁷⁸, rabbit serum containing Sigma Receptor (σ₁R) antibodies (kind gift from Dr. Arnold Rhouho) or Living Colors^® A. V. mouse monoclonal antibody against EYFP (JL-8) (Clontech) to a final concentration of 2 µg. IP and total lysates were subjected to electrophoresis on 10% NEXT GEL^® polyacrylamide gels (VWR Internationals) and transferred to 0.2 µm PVDF membranes. DAT, σ₁R, and EYFP were detected using mouse monoclonal antibody 16 (mAB), mouse monoclonal σ₁R (B-5) antibody, and rabbit polyclonal Living Colors^® A. V. anti-EYFP antibody (Clontech). Bound 1° antibodies were detected with polyclonal goat anti-mouse antibodies (A3562, Sigma Aldrich) (DAT, σ₁R) or goat-anti-rabbit polyclonal antibodies (EYFP) linked to alkaline phosphatase (A6387, Sigma Aldrich) or horseradish peroxidase (Millipore). Rabbit polyclonal Drosophila SERT antibodies (13-A) were used as the unrelated antibody control (Alpha Diagnostics Inc.). The mock control was parental HEK-293 cells that have endogenous σ₁R but do not express DAT. Images have been cropped for presentation. Full size images are presented in Supplementary Figs. ⁹ and ¹⁰.