Figure 4.

Incubation of renal epithelial cells with 2 different FLCs (κ2 and λ3) increase nuclear NF-κB activity and promote MCP-1 production in an Src kinase–dependent fashion. (A) Nuclear lysates were obtained from HK-2 cells after 4 and 24 hours of incubation in medium containing 2 different FLCs (κ2 and λ3). Nuclear NF-κB activity was quantified using a filter plate assay, which consisted initially of incubating nuclear extracts with a biotinylated NF-κB–specific DNA-binding sequence. This NF-κB DNA complex was captured on a filter plate to remove the unbound probes and was then denatured and hybridized onto precoated microwells. Bound NF-κB DNA complexes were detected with streptavidin–horseradish peroxidase and quantified using a luminometer. Results, expressed in relative light units, showed an increase in nuclear NF-κB activity at 4 and 24 hours of incubation of renal epithelial cells with κ2 and λ3 FLCs compared with medium alone. The addition of PP2 to the medium inhibited the FLC-induced increase in activity. *P < .05 compared with contemporaneous samples incubated in medium alone, PP2, and corresponding FLCs and PP2; n = 6-12 experiments in each group. (B) Increase in production of MCP-1 after overnight incubation of renal epithelial cells with the κ2 and λ3 FLCs; the addition of PP2 prevented this FLC-induced increase in MCP-1. *P < .05 compared with control and samples treated with PP2 and corresponding FLCs; n = 6 experiments in each group