Western blot of CXCL7 peptides. (A) Western analyses of culture media from CD14⁺ and HS27a cells. Culture media were collected, and Western analyses were performed for CXCL7 peptides. Lanes 1 and 2 represent positive controls NAP-2 and PBP, respectively. Culture conditions for the other lanes are as follows: CD14⁺ cells cocultured with HS27a cells for 3 days (lane 3), CD14⁺ cells cocultured with HS27a cells for 6 days (lane 4), CD14⁺ cells cultured alone for 6 days (lane 5), and HS27a cells cultured alone for 6 days (lane 6). At day 3, the CXCL7 peptides secreted are of comparable size to PBP, whereas by day 6, two distinct peptides are detected, PBP and NAP-2. (B) Western analyses of CD14⁺ coculture with HS5 cells. As in panel 3A, lanes 1 and 2 show NAP-2 and PBP. Culture conditions for the rest of the lanes are as follows: CD14⁺ cells cocultured with HS5 cells for 3 days (lane 3); CD14⁺ cells cocultured with HS5 cells for 6 days (lane 4); CD14⁺ cells cultured alone for 6 days (lane 5); and HS5 cells cultured alone for 6 days (lane 6). Here, only the PBP peptide is detected at days 3 and 6 of coculture. (C) CXCL7 peptides secreted by PBMCs in response to LPS stimulation. PBMCs were stimulated by 1 μg/mL LPS for 24 hours. Positive controls are β-TG, recombinant NAP-2, and recombinant PBP (lanes 1, 2, and 7). Conditions for the rest of the lanes are as follows: media from PBMCs cultured with LPS (lane 3), cell extract from PBMCs with LPS (lane 4), media and cell extract from PBMCs cultured without LPS (lanes 5 and 6). PBP is detectable in the culture media stimulated by LPS; both PBP and β-TG are detectable in the cellular extract. No CXCL7 peptides are seen when no LPS is added.