Protein S production by growing and growth-arrested human vascular smooth muscle cells (HVSMCs).

Protein S production was assessed in conditioned media. Two cell culture conditions were used: growth-arrested (GA) and growing (GR) cells. Growth-arrested cells were obtained by transferring 90% confluent cultures into serum-free Dulbecco's modified Eagle's medium (DMEM) for 48 hours prior to the commencement of media conditioning. Growing cells, were subconfluent cultures (60% confluency) that were placed in a defined growth promoting serum-free medium at the start of media conditioning. Conditioned media were collected after 24 hours (lanes 1, 4), 48 hours (lanes 2, 5), and 72 hours (lanes 3, 6) and were analyzed by Western blotting using a rabbit anti-protein S antibody. Purified 1 ng protein S (pS) and 50 ng recombinant human growth arrest specific protein 6 (G6) were included as controls.