Rabbit anti ha primary antibody

HEK293A cells were seeded in 6-well plates and transfected with 4 μg plasmid containing GPCR-HA and FLAG-RAMP at a ratio of 1:1. After 24 h, cells were collected and reseeded in 96-well plates until the cells reached 50%–70% confluences. They were washed with PBS before fixation with 4% paraformaldehyde for 15 min. Then they were washed three more times and blocked with 5% BSA plus 0.1% Triton X-100 for 1 h at room temperature (RT). Rabbit anti-HA primary antibody (Cell Signaling Technology, Danvers, MA, USA; 1:500) and mouse anti-FLAG primary antibody (Sigma–Aldrich, St. Louis, MO, USA; 1:300) were diluted with incubation buffer (PBS supplemented with 5% BSA) for 1 h followed by 3-time wash. Cells were reacted with 100 μL interaction buffer containing donkey anti-rabbit Alexa 488-conjugated secondary antibody and donkey anti-mouse Alexa 647-conjugated secondary antibody (Invitrogen; diluted 1:1000) at RT for 1 h in the dark. After final washing, nuclei were stained with Hoechst 33,258 for 5 min. Cells were imaged using high-resolution microscope DeltaVision™ Ultra (GE Healthcare, Boston, USA).