Figure 1. The kinin B1 receptor is functionally expressed in Tm5 melanoma cells.
(A) Expression levels of the kinin B1 and B2 receptors, carboxypeptidase M (CPM) and cyclophilin B as a housekeeping gene in melanocytes (melan-a) and melanoma cells (Tm5) evaluated by RT-sqPCR. cDNA from mice hearts was used as a positive control, and water was used as a negative control. (B–C) Kinin B1 receptor is present at protein level in Tm5 cells. Agonist stimulation of the cells has no effect in B1 receptor protein level, however a significant increase in mRNA level was detected. The activity of the B1 receptor in Tm5 cells was accessed by western blotting for ERK phosphorylation (D) and confocal microscopy using FLUO3-AM to evaluate intracellular calcium mobilization (E) after stimulation with the agonist DABK and blockage by the antagonist DLBK. All results are representative or quantification of 3–5 independent experiments. Data are expressed as the mean ± SEM; * p<0.05, with respect to non-stimulated cells; DABK: desArg9-bradykinin; DLBK: desArg9-[Leu8]-bradykinin.