Fig. 3.

(A) PMA causes an increase in secreted ANGPTL4 protein. HASM cells were grown for 24 h in serum-free medium and then treated for 16 h with 30 nM PMA or vehicle (C). Conditioned medium was collected, concentrated, and examined by Western blot analysis using an anti-ANGPTL4 antibody. (B) HASM cells were treated with actinomycin D (2.5 μg/ml) for 30 min prior to the addition of PMA (30 nM) or DMSO and 6 h later, total RNA was isolated and analyzed by Northern Blot analysis for ANGPTL4 expression. (C) Effect of PMA on the stability of ANGPTL4 mRNA. HASM cells were treated with 30 nM PMA or DMSO. After 4 h treatment, actinomycin D (2.5 μg/ml) was added and at the times indicated cells were collected for RNA isolation. Total RNA was isolated from PMA-treated cells and because DMSO-treated cells express very low levels of ANGPTL4, poly(A)⁺ RNA was isolated from vehicle-treated cells. ANGPTL4 and α-tubulin mRNA expression were examined by Northern Blot analysis. The level of ANGPTL4 mRNA (relative to the level of α-tubulin mRNA) was calculated and the results plotted as the percentage of the RNA level present at time 0 of actinomycin D addition. Data shown is representative of two independent experiments.