Fig. 8.

(A) mAPJ-HEK293 cells were pre-incubated in the presence or absence of [Pyr¹]apelin-13 (100 nM, 2 h), washed, incubated in fresh medium for 0–1 h and then stimulated in the presence or absence of [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 ([Pyr¹]apelin-13-induced ERK1/2 signalling in cells initially exposed to vehicle control and designated as “max”). In (B–D) mAPJ HEK293 cell lines were transfected with GRK^DNM, DYN^DNM or βARR^DNM cDNAs respectively before preincubation with or without [Pyr¹]apelin-13. Data shown are mean ± SEM, of at least three separate experiments, each with triplicate wells and triplicate fields within wells. ***p < 0.001, comparing stimulations to max conditions, analysed by one-way ANOVA and Dunnett's multiple comparison post hoc tests.