Fig. 7.

Recovery of APJ levels after agonist removal. HA-mAPJ-HEK293 cells were incubated in the presence or absence of [Pyr¹]apelin-13 (100 nM) for 2 h, washed, then incubated in fresh medium for 0–6 h. Cells were fixed, stained and imaged for determination of either (A) cell surface or (B) whole cell HA-mAPJ intensity using anti-HA antibody. The value determined with no primary antibody present was designated as background and was subtracted from raw data to give HA-mAPJ intensity in arbitrary fluorescence units (AFU) and then normalized to a percentage of vehicle control. Cell surface and whole cell AFU values were used to determine the PCSE (C). (D) shows recovery from the effect of [Pyr¹]apelin-13 on HA-mAPJ inclusion count. HA-mAPJ-HEK293 cells were pre-treated with anti-HA antibody (1:100; 1 h, 37 °C/5% CO₂), washed, incubated in the presence or absence of [Pyr¹]apelin-13 (100 nM) for 2 h to internalize APJ, washed, then incubated in fresh medium for 0–6 h. Data shown are mean ± SEM, of three separate experiments, each with triplicate wells and triplicate fields within wells. *p < 0.05 and **p < 0.01, comparing stimulations to basal conditions, analysed by one-way ANOVA and Dunnett's multiple comparison post hoc tests.