Secretion of GRP78 and MMP-1 in conditioned media of breast cancer cells. Breast cancer cells MCF-7 and MDA-MB-231 as well as the control MCF-10A were all cultured overnight in EMEM containing 10% fetal bovine serum. The conditioned media were collected, dialyzed overnight at 4°C in 50 mM Tris-HCl, pH 7.0, and concentrated approximately 1000-fold over Aquacide II™. Equal amount of protein from each sample was analyzed by western blotting. (A) GRP78; lane 1 = media from MCF-10A cells, lane 2 = media from MCF-7 cells, lane 3 = media from MDA-MB-231 cells, lane 4 = EMEM with 10% serum, lane 5 = MDA-MB-231 whole cell lysate; (B) MMP-1; lane 1 = EMEM with 0% fetal bovine serum, lane 2 = EMEM with 10% fetal bovine serum, lane 3 = fetal bovine serum from stock. The error bars represent the standard error (Mean ± SE) for n = 3. P-values are from the Student t-test (** = P < 0.005; *** = P > 0.00005; **** = P > 0.000005).