D24h9 rabbit

Western blot was performed as previously described^{58 (link)}. Briefly, denatured proteins were resolved in 12% SDS-PAGE gels, transferred to nitrocellulose membranes, and immunoblotted with antibodies against each primary antibody as bellows; anti-flag antibody (1:1000 dilution, rabbit F7425 Sigma-Aldrich), anti-HA antibody (1:1000 dilution 16B12 mouse, 901514 Biolegend), anti-myc antibody (1:1000 dilution; 9E10 mouse, sc-40; Santa Cruz Biotechnology), anti-ß-arrestin1/2 antibody (1:1000 dilution; D24H9 rabbit; Cell signaling), Immunoreactivity was revealed using secondary antibodies coupled to 680 or 800 nm fluorophores (LI-COR Biosciences, Lincoln, NE, USA), and readings were performed with the Odyssey LI-COR infrared fluorescent scanner (LICOR Biosciences).

The expression of HA-tagged (N-terminus) GPR61, GPR62 or GPR135 receptors were detected by immunofluorescence as previously described^{53 (link)}. Briefly HEK293T cells transiently expressing HA- or myc-tagged GPR61, GPR62 or GPR135 (1 ug DNA) were seeded onto sterile poly-L-lysine–coated 24-well glass 1 day after transfection. Next day, with or without Transferrin-Alexa555 (Molecular Probes; T-35352) treatment (5 μg/ml for 30 minuites in 37 °C after washing by serum free medium), cells were fixed with a 4% fresh paraformaldehyde solution (in PBS) for 20 min. With or without 5-min permeabilization step in 0.2% Triton X-100 (in PBS), cells were blocked for 1 hour with 5% BSA (in PBS). Polyclonal anti-HA antibody (dilution 1:500, rabbit, Cell Signaling Technology), monoclonal anti-myc antibody (dilution 1:500, 9E10 mouse, sc-40; Santa Cruz Biotechnology), or anti-ß-arrestin1/2 antibody (1:1000 dilution; D24H9 rabbit; Cell signaling) was applied followed by secondary fluorescein isothiocyanate-tagged anti-rabbit or anti-mouse antibodies (dilution 1:500, Jackson Immunotech 111-095-003). Cells were examined by confocal microscopy (Leica, Germany).