Figure 4.

Overexpression of GEF1 or its activated Rho substrates enhances release of unprocessed and partially processed secretory cargo. AtT-20 cells were nucleofected with the indicated plasmids. Media (from 24 to 48 h after transfection) and cells were collected and analyzed by immunoblotting. (A) Schematic of POMC processing. PC1 cleavage sites and antibody specificity are indicated. (B) Representative blots from control-KalGEF1 and ND/AA-transfected cells. Two volumes of lysate and one volume of medium (2× and 1×) were loaded to yield comparable signal intensities; cells shown release this amount of POMC products in 12 h. Although POMC product signals in cells and media for each vector are comparable, Control, Kal-GEF1 and ND/AA cells were not normalized and are not comparable. (C) Secretion indices from blots obtained as in B were determined by dividing the signal intensities of the media by those of the corresponding lysates for each POMC product, after normalization to the volumes loaded. Indices, normalized to the corresponding value in controls, are expressed as means ± SEM; significant differences are shown (paired t test; n = 6). (D) Cells nucleofected with the indicated plasmids were treated and processed as in B; n = 6. (E) Cells were nucleofected with the indicated plasmids (empty pCMS EGFP vector was used as control) as in B and C. Secretagogue challenge was for 30 min with 2 mM BaCl₂. Release of the 16-kDa product was assessed by immunoblotting and expressed as percent of cell content (means ± SEM; n = 3).