Fig. 2.

Increased expressions of cytokines and chemokines and NF-κB activity in IKK1-deficient macrophages. (A) Expression of TNF-α, MIP1α, MCP1, and Cox2 in response to LPS stimulation. IKK1 WT and mutant ELDM were stimulated with LPS (100 ng/ml) for 2, 4, and 6 h, and total RNA was extracted with TRIzol. After cDNA synthesis, Q-PCR was performed. The data were normalized with the expression of cyclophylin A. Data shown are a representative result from one of three independent experiments. (B) Proinflammatory cytokine level in the culture supernatant was determined by quantitative ELISA. Confluent ELDM were treated with LPS (100 ng/ml) for 6, 12, and 22 h, and their supernatants were collected and spun to remove cell debris and stored at –80°C for ELISA analysis. Total proteins were extracted from the ELDM, and their concentrations were determined and used to normalize the ELISA results. Data are the average from triplicates.(C) IKK1 WT and mutant ELDM were infected with lentiviruses containing the κB-luciferase reporter construct. Two days later, cells were treated with various NF-κB stimuli as indicated for 4 h after being serum-starved (DMEM containing 0.5% FCS) for 4 h. The luciferase assay was performed by using protein lysis. Results were normalized by basal activity of cells untreated and averaged from triplicates. (D) A similar analysis as in C was done in mouse embryonic fibroblasts either WT or deficient of IKK1/2, p65, and p50.