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Fig. 5. Erk1/2 phosphorylation is sensitive to B. pertussis toxin and is not achieved by triggering the CCR7 mutants MT1 and DNY. (A) MT1 triggering by CCL19 does not lead to Erk1/2 phosphorylation. HEK293 cells stably expressing CCR7 or the C-terminal truncations MT1-MT3 were incubated with 2 µg/ml CCL19 for 5 minutes. Western blots derived from total cell lysates were probed with a monoclonal antibody specific for phosphorylated Erk1/2 (pErk-1/2). The nitrocellulose membrane was stripped and reprobed with a polyclonal anti-total Erk2 (tErk-2) antibody as a loading control. (B) PTx treatment impairs CCR7 mediated Erk1/2 activation. HEK293 cells stably transfected with wild-type CCR7 were pretreated or not with 100 ng/ml PTx for 2 hours followed by stimulation with 2 µg/ml CCL19 for 5 minutes. Erk1/2 phosphorylation was analyzed by western blotting as in A. (C) DNY mutation abolishes chemokine-mediated Erk1/2 activation. 300-19 cells stably expressing wild-type CCR7 and the DNY mutant were left untreated or incubated with 2 µg/ml CCL19 for 5 minutes and Erk1/2 phosphorylation was analyzed as above. One out of at least three experiments with similar results is shown.

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