Regulated secretion of GH and vWF from HUVEC. (A) HUVEC were infected with IGrAd (1 and 2) or GHrAd (3 and 4) and labeled with [³⁵S]-methionine/cysteine for 3 days. Labeling medium (1 and 3) and chase supernatants (2 and 4) were then subjected to immunoprecipitation and SDS-PAGE as described in Materials and Methods, indicating sizes of approximately 22 and 34 kD, respectively. (B and C) HUVEC were infected and labeled with [³⁵S]-methionine/cysteine, and after 16 hours of chase with unlabeled medium, the cells were treated for 30 minutes with control buffer or 100 nmol/L PMA. Supernatants were not subjected to immunoprecipitation before loading onto gels. (B) Treatment supernatants were analyzed by SDS-PAGE and phosphorimaging. Shown is a gel representative of 3 similar experiments, with mock-infected HUVEC treated with control buffer (1) or PMA (2) and GHrAd-infected HUVEC treated with control buffer (3) or PMA (4) (m-vWF, mature vWF; propeptide, the cleaved vWF propeptide). (C) Shown are chase and treatment supernatants analyzed by SDS-PAGE and densitometry, representative of 3 different experiments; chase supernatants from IGrAd (1 and 2) and GHrAd (3 and 4), control treatment supernatants from IGrAd (5) and GHrAd (7), and PMA treatment supernatants from IGrAd (6) and GHrAd (8). The relative sorting efficiency or GH compared with vWF was calculated from densitometric analysis of pro-vWF and GH in lane 4 and m-vWF and GH in lane 8.