Anti d2r antibody

Co-immunoprecipitation was performed as previously described (Hasbi et al., 2014 (link); 2018 ). Protein homogenates (300 μg) obtained from tissue punches of different regions of rat brain were incubated with anti-D2R antibody (Alomone Laboratories) at 4°C overnight under gentle rotation. After adding 40–50 μl of protein G/A, the mixture was further incubated for 1 h. After 3 washes with PBS-Tween, Laemmli buffer was added, and the immunoprecipitate was incubated for 5 min at 95°C. Proteins were resolved by electrophoresis on 10% polyacrylamide gels under denaturing conditions (SDS-PAGE) and transferred onto nitrocellulose or PVDF membranes (Bio-Rad Laboratories, Hercules, CA, USA) using a semidry transfer system (Invitrogen, Carlsbad, CA, USA). Membranes were incubated in PBS-Tween (PBS-T)/10% nonfat milk for 1 h. After 3 washes, membranes were incubated with PBS-T/5% nonfat milk containing the anti-D1R antibody raised in rats (Sigma, D2944). Membranes were washed once in PBS-T and 2 times in PBS (10 min each) and incubated with the appropriate polyclonal secondary antibody for 2 h. After 3 washes as indicated above, signal detection was performed using a Li-Cor instrument (Odyssey).