Ab78021

PLA was used for the in situ detection of the interaction between D1 and D2 receptors and also between σ1Rs and IP₃R1s. The assay was performed in a humidified chamber at 37 °C according to the instructions of the manufacturer (Olink Bioscience, Sweden). For this method, following antibodies were used: rabbit polyclonal antibody to OPRS1 (σ1R; AB_881796, Abcam, UK), mouse monoclonal antibody to IP₃R1 (AB_212025, Calbiochem, Merck Biosciences, Germany), mouse monoclonal antibody to dopamine receptor D1 (SG2-D1α, ab78021, Abcam, UK), and rabbit polyclonal antibody to dopamine receptor D2 (ab21218, Abcam, UK).

Membrane proteins were transferred from BN-PAGE to PVDF membranes. After blocking of membranes for 1 h with 10 % non-fat dry milk in 0.1% TBST (100 mM Tris–HCL, 150 mM NaCl, pH 7.5, 0.1% Tween 20), membranes were incubated with primary antibodies D1R (diluted 1:5000, Abcam-ab78021, Cambridge, UK), D2R (diluted 1:5000, Abcam-ab21218, Cambridge, UK), D3R (diluted 1:5000, Abcam- ab42114, Cambridge, UK), pDAT (diluted 1:5000, DAT Thr⁵³, Phosphosolutions-p435-53, Aurora, United States) and DAT (diluted 1:5000, Abcam- ab111468, Cambridge, UK) and detected with horseradish peroxidase-conjugated anti-rabbit IgG (diluted 1:10000, Abcam- ab6721, Cambridge, UK). Membranes were developed with the Bio-Rad Clarity^TM Western ECL Substrate. Arbitrary optical densities of immunoreactive bands were measured by the Image J software program¹. After developing, total protein staining was done on PVDF membranes as previously described for loading control.