Expression and functional analysis of wild-type and variant D304N TxA₂R expressed in CHO-K1 cells. (A) CHO-K1 cells were transfected with expression constructs containing wild-type (WT TxA₂R) or variant D304N TxA₂R (D304N TxA₂R). Expression levels of TxA₂R in CHO cell lysates were determined by ELISA using anti-FLAG M2 monoclonal antibody. Data represent means ± SEM of 3 independent experiments and are reported as arbitrary units (AU) relative to expression level of the wild-type TxA₂R (designated as 1AU). (B) Transfected CHO-K1 expressing wild-type and variant D304N TxA₂R were also studied by immunofluorescence microscopy using an anti-FLAG M2 monoclonal antibody. The images are representative of > 20 high power fields. (C) Changes in cytoplasmic calcium concentration [Ca²⁺]_i were measured by preincubating washed transfected CHO-K1 cells with fura-2/AM (3μM) before addition of U46619 (0.001-10μM). Data are expressed as peak calcium response quantified as described previously²⁰  and represent the means ± SEM of 3 independent experiments.