Secretion of MMP-9 protein. (A) CD14+ cells (0.1 × 106 cells) were cultured for 5 days in control medium or conditioned medium from HS-5 (lanes 1 or 2, respectively). Culture media were harvested. The media were immunoprecipitated, proteins were denatured, run on SDS-PAGE, and transferred onto a nitrocellulose membrane. The membrane was incubated with polyclonal anti–MMP-9 antibodies, followed by fluorescent-labeled secondary antibodies. As a control, fresh medium was used as a mock immunoprecipitation (lane 3). The conditioned medium from HS-5 cells was also negative (data not shown). An arrowhead indicates the position of MMP-9 protein. Immunoglobulin heavy and light chains (∼60 kDa and ∼25 kDa) are stained in all 3 lanes. This is a representative blot of 3 similar experiments. MW indicates a lane for prestained molecular weight markers. (B) Fluorescence intensity of MMP-9 protein bands in lanes 1 and 2 was determined by Odyssey scanner (Control and HS5CM, respectively). II indicates integrated intensity.
