Overexpression of PTH1R receptor in a HEK293 cell model HEK293 cells (p31) were plated in a 6-well plate at a density of 500,000 cells/well and incubated at 37 °C/5% CO2 to 70-80% confluency. PTH1R overexpression was achieved by transfection with the PTH1R plasmid. Briefly, Lipofectamine 3000 was prepared by mixing 10 μL of the reagent with 250 μL of OptiMEM. PTH1R (2.7 μL, 0.9 mg/mL) was diluted in 125 μL OptiMEM to which 5 μL of P3000 reagent was added.