Figure 1
Expression and [3H]-MLT and 2-[125I]-MLT binding to HA-GPR61, HA-GPR62 and HA-GPR135 expressed in HEK293T cells. (A) The expression of HA-tagged (N-terminal) GPR61, GPR62 and GPR135 at the cell surface of intact HEK293T cells was detected by confocal microscopy with anti-HA antibodies. (B) Expression of HA-GPR61, HA-GPR62 and HA-GPR135 in crude membranes prepared from HEK293T cells was monitored by western blot. Different denaturating conditions were applied: RT; room temperature overnight, 55 °C 15 min, or 95 °C 10 min, all in the presence of DTT. (C,D) Binding of [3H]-MLT (C) and 2-[125I]-MLT (D) to crude membrane prepared from HEK293 cells expressing HA-tagged or untagged GPR61, GPR62 or GPR135. Expression of HA-tagged receptors was verified by western blot (insets). Flag-MT1 was used as positive control. Representative results are shown for panels A and B; similar results were obtained in three additional experiments. Data in panels C and D represent the mean ± SEM of three independent experiments performed in duplicates. ***P < 0.001.
