Figure 6.

Inhibition of endogenous GEF1 activity blunts release of secretory cargo. (A) AtT-20 cells were incubated with medium containing vehicle (DMSO) or 100 μM NPPD for 3 h. Cells and media were analyzed as described in Figure 4. Blots of lysates (C1, C2) and media (M1, M2) from two samples for each treatment are shown (top panel). Secretion indices were determined (means ± SEM), middle panel. From the same blots, the signal intensity of the cellular 16-kDa product was normalized to the corresponding γ-adaptin signal (means ± SEM; bottom panel). (B) AtT-20 cells were pretreated for 2.5 h in CSFM with 100 μM NPPD or the same amount of vehicle (DMSO) and then challenged with 2 mM BaCl₂ for 30 min (with DMSO or NPPD). A representative immunoblot from media is shown (top panel). The amount of mature 16-kDa product secreted was determined (means ± SEM; bottom panel). (C) AtT-20 cells were cotransfected by nucleofection with signalGFP vector and control vector pEAK10 or pEAK10 vector encoding Kal-GEF1. Cells were either treated with DMSO or NPPD (100 μM) for 2 h. Cell lysates and media separated by SDS-PAGE were probed for GFP; the signals were densitized (n = 3).