R37116

SH-SY5Y cells were seeded on glass coverslips and cultured for 24 h and fixed with 4% paraformaldehyde for 15 min. After washing with PBS, the cells were first incubated with 50 mM PBS containing 10% normal goat serum and 0.5% TritonX-100 at room temperature for 2 h to block non-specific binding and this was followed by incubation with rabbit anti-GPER (1:500, Abcam, Cat# ab39742, RRID:AB_1141090) or rabbit anti-MOR (1:500, Novus, Cat# NBP1-31180, RRID:AB_2251717) at 4°C overnight. The cells were rinsed with PBS for four times and were then incubated with goat anti-rabbit Alexa fluor 568 (1:1000; Molecular Probes-Invitrogen, Cat# A-11077, RRID:AB_141874) or 488 (1:1000; Molecular Probes-Invitrogen, Cat# R37116, RRID:AB_2556544) secondary antibody at room temperature for 1.5 h. GPER or MOR were counter-stained with a nuclear marker DAPI (1: 1000, Thermo Fisher Scientific, Cat# PA5-62248, RRID:AB_2645277) at room temperature for 10 min. The coverslips were mounted on glass slides and the cells were viewed under the fluorescent microscope (Leica DM2500, Leica Microsystems Limited).

HepG2 cells were plated at 150,000 cells per well on acid-washed and sterilized glass coverslips in a 12-well plate. Cells were stimulated as described before and then washed at 1, 6, 12, and 24 h in cold PBS (Gibco) and fixed for 10 min in 4% paraformaldehyde (AAJ19943K2, Thermo Fisher Scientific). Cells were blocked in 10% BSA and permeabilized with Triton X-100. 1:600 anti-ATP7B (ab124973, Abcam) and 1:600 anti-TGN46 (GTX74290, GeneTex) antibodies were used to stain the copper transporter and trans-Golgi network marker, respectively, and anti-rabbit IgG AlexaFluor 488 (R37116, Invitrogen) and anti-sheep IgG AlexaFluor 647 (A21448, Invitrogen) were used as secondary antibodies to fluorescently label the proteins. A DAPI dye (R37606, Invitrogen) was used to stain the nucleus, and coverslips were sealed to glass slides along with Prolong Gold Antifade Mountant (P36930, Invitrogen). Fixed and mounted cells were imaged using a laser scanning confocal microscope (Olympus FluoView FV1000) using a ×60 oil immersion lens at the UC Davis Molecular and Cellular Biology Light Microscopy Imaging Facility.