Fig. 3.

Desensitization of [Pyr¹]apelin-13-induced ERK1/2 activation in mAPJ-HEK293. mAPJ-HEK293 cells were pre-incubated with PBS or [Pyr¹]apelin-13 (100 nM, 2 h), washed, and immediately stimulated in the presence of (A) adrenaline (1 μM) (B) EGF (100 ng/ml) or (C) [Pyr¹]apelin-13 (100 nM) for 5 min. Cells were fixed, stained and imaged for determination of whole cell ppERK1/2 intensity using anti-ppERK1/2 antibody. The value determined with no primary antibody present was designated as background and was subtracted from raw data to give ppERK1/2 intensity in arbitrary fluorescence units (AFU) and then normalized to a percentage of vehicle control. Data shown are mean ± SEM, of at least three separate experiments, each with triplicate wells and triplicate fields within wells. ***p < 0.001, analysed by one-way ANOVA and Dunnett’s multiple comparison post hoc tests. ns = no statistical significant difference.