Figure 2.

Pitstop2 and BimI blocked PACAP-mediated ERK activation at varying PACAP concentrations. HEK PAC1 receptor-EGFP cells were pretreated with 20 μM Pitstop 2 (A) or 15 μM BimI (B) for 15 min before the addition of PACAP (5 - 20 nM) for 15 min. Pitstop and Bim I inhibited ERK activation at all PACAP peptide concentrations tested, suggesting that receptor internalization/endosomal signaling occurred even at the low peptide concentrations. Even at 1 nM PACAP, ERK activation was more sensitive to receptor internalization/endosome-mediated mechanisms than PKC signaling. Representative Western blots for each treatment shown in replicate. *, significantly different from untreated control (CTL); +, different from sample at same PACAP concentration. Data represent mean ± SEM, n = 3.