The Immunomodulatory Effects of Albumin In Vitro and In Vivo
Figure 4
Albumin preconditioning attenuates LPS-mediated NF-κB activation. (a) NF-κB luciferase experiments. RAW264.7 peritoneal macrophages were transiently transfected with a 3x NF -κB/luc reporter plasmid. Cells were allowed to recover overnight and were then preconditioned with BSA (1 mg/mL) for 18 h prior to a subsequent treatment with LPS (10 μg/mL). LPS treatment resulted in a significant increase in NF-κB promoter activation, which was significantly inhibited by albumin preconditioning. All experiments were performed in triplicate with 3 wells per condition (* compared to control; # compared to LPS alone). (b) EMSA. RAW264.7 peritoneal macrophages were preconditioned with BSA (1 mg/mL) for 18 h prior to a subsequent treatment with LPS (10 μg/mL). Nuclear protein was harvested at 30 min after LPS and EMSA were performed. LPS treatment resulted in a significant increase in NF-κB binding (Lane 2) compared to either control (Lane 1) or albumin preconditioning alone (Lane 3). Albumin preconditioning abrogated NF-κB binding (Lane 4) compared to LPS alone. Lane 5 (LPS treatment, cold competitor), Lane 6 (LPS treatment, p65 supershift), Lane 7 (LPS treatment, p50 supershift), Lane 8 (Alb + LPS, cold competitor), Lane 9 (Alb + LPS, p65 supershift), and Lane 10 (Alb + LPS, p50 supershift) were performed as additional controls to demonstrate specificity and the nature of the NF-κB. The EMSA shown is representative of 3 separate experiments, all with similar results.
