BCA-1 enhances receptor internalization and ERK phosphorylation in CCR7-transfected cells. BCA-1 enhances SLC- (A,B,E) and ELC- (C,D,E) induced CCR7 internalization. The histograms represent the fluorescence intensity of CCR7 in transfected cells unstimulated (light gray curves), stimulated with 300 nM SLC (A) or 100 nM ELC (C) (dark gray curves), or sequentially stimulated with 1 μM BCA-1 and SLC or ELC (bold line), as analyzed by flow cytometry. One representative experiment of 3 is shown. Time course of CCR7 internalization induced by 300 nM SLC (B) or 100 nM ELC (D) in the presence (closed symbols) or absence (open symbols) of 1 μM BCA-1. One of 2 independent experiments is shown. (E) CCR7 expression in cells stimulated for 30 minutes with 300 nM SLC or 100 nM ELC in the presence (filled symbols) or absence (open symbols) of 1 μM BCA-1. Columns represent mean ± SEM of 5 independent experiments. Differences between receptor expression after stimulation with the agonist or with the agonist plus BCA-1 are statistically significant. (F-G) ERK phosphorylation mediated by 100 nM SLC (F) or 1 μM BCA-1 (G) alone after times indicated. (H) ERK phosphorylation by a suboptimal dose (1 nM) of SLC and its synergistic increase in the presence of 1 μM BCA-1 after times indicated. pERK designates phosphorylated, and ERK-2 total ERK, respectively. One representative experiment of 3 is shown. (I) Densitometric analysis of the bands corresponding to phosphorylated ERK induced by SLC (1 nM; □) or SLC (1 nM) plus BCA-1 (1 μM; ▪). Values were normalized against their matching total ERK values, and the arbitrary value of 1 was given to the experiments obtained with unstimulated cells (ns). Mean ± SD of 3 independent experiments is shown.