Figure 5.

Conjugation of CGRP_8-37 to /D∆H_C-CS yielded /D-CGRP_8-37 which cleaved VAMP1 and inhibited depolarization evoked substance P release from cultured DRGs. (A) Schematic of /D-CGRP_8-37. (B, C) DTT reduced /D-CGRP_8-37 and /D∆H_C-CS samples were analyzed by SDS-PAGE followed by Coomassie staining (B) or by Western blotting with a rabbit anti-CGRP antibody (1:10,000) (C). Uncropped original images are shown in Figure S4. (D) Depolarization of cultured DRGs stimulated the release of substance P approximately 5-fold over basal release (LK, low potassium; HK, high potassium). (E) Representative Western blots showing the cleavage of VAMP1 by /D-CGRP_8-37 compared to /D∆H_C-CS. Syntaxin1 and SNAP-25 were probed as loading control. (F) Intact VAMP1 remaining after incubating with 100 nM or 200 nM of either protein was calculated by expressing the ratio (intensity of VAMP1/the corresponding internal loading control (Syntaxin1)) as a percentage of the toxin free control sample. (G) /D-CGRP_8-37 but not/D∆H_C-CS attenuated the K⁺-evoked substance P release when compared to the toxin-free sample. Data plotted in (D,F,G) are mean ± S.E.M. (n = 3). ns: non-significant, * p < 0.05 and *** p < 0.001.