Upon binding of PTH/teriparatide to the PTH1R receptor a predominant activation of the Gαs signalling pathway is observed, which results in a series of downstream signalling such as production of cAMP but also phosphorylation of Erk1/2 (also known as p44/42 MAPK), which is exploited in this assay . An hour prior to exposure of the HEK293 and HEK293-PTH1R cells to teriparatide, serum was removed from the cell culture, to reduce baseline phosphorylation . Untransfected HEK293 and HEK293-PTH1R cells were exposed to teriparatide (10 nM) for 5 -45 min covering both the early rapid activation and the later sustained activation phase . Following incubation, each treatment group was lysed and analysed by Western blotting to measure total Erk and phosphorylated Erk levels. Densitometric band analysis of The figure demonstrates that baseline phosphorylation of Erk in the HEK293-PTH1R cells is higher than that of HEK293 cells and that the temporal increase in Erk phosphorylation is higher in HEK293-PTH1R cells due to PTH1R over-expression.