Figure S1. Elevated Intracellular Free Ca²⁺ Elicited by FLRF in HEK Cells Expressing mrgA1

(A), (B), (E), and (F) illustrate Fura-2 fluorescence at 340 nm (A), (E) and 380 nm (B), (F) in HEK-Ga₁₅ cells expressing an mrgA1-GFP fusion protein (A-D) or GFP alone (E-H). The images were taken 2 minutes after the addition of 1 mM of FLRFamide. The perinuclear, punctate distribution of mrgA1-GFP revealed by intrinsic GFP fluorescence (D, arrowheads) is characteristic of the ER-Golgi network, indicating membrane integration and intracellular transport of the receptors. In contrast, the control GFP protein is cytoplasmic (H). The intracellular Ca²⁺ ([Ca²⁺]_i) release was determined from the FURA-2 340 nm/380 nm emission ratio (C,G). Note that mrgA1-expressing cells (but not surrounding untransfected cells) show an elevated ratio of Fura-2 fluorescence at 340/380 nm (C, arrowheads), indicating an increase in [Ca²⁺]_i. In contrast, no such elevation is observed in control GFP-expressing cells (G). The elevated 340/380 fluorescence seen in mrgA1-expressing cells was dependent on the addition of ligand (not shown).