Galr1-EGFP Fusion Protein Expression

Gal₁R-EGFP vectors were made by reverse transcribing the open reading frame of the rat Galr1 gene, followed by PCR amplification using the primers as shown in SI Table 3. PCR products were ligated into a pEGFP-NI vector (Clontech) at its HindIII/SacII and Hind/KpnI restriction sites to generate Galr1-EGFP expression vectors. Rat pheochromocytoma cells (PC12) (1,000,000 cells/plate) were transfected with Galr1-Egfp (1 µg plasmid DNA) using Effectene Transfection Reagent (Qiagen), as described earlier^{46 (link)}. Stably transfected cells were selected by growing them in the presence of geneticin (G418, Sigma) at a concentration of 800 µg/ml. PC12 cells were sub-cultured and used for 10–15 passages. For Gal₁R-EGFP internalization, cells were seeded on 13-mm glass coverslips (10–20,000 cells/well in 24-well plates) previously coated with poly-D-lysine (Sigma), and pharmacologically probed 72 h after plating. Galanin was superfused at a concentration of 100 nM. Cells were photographed using differential interference contrast (DIC) microscopy (DMLFSA, Leica).