Development of the upstream model and full model fits. (A) The upstream model with A20 inhibition at two points was modified to include nonlinear IKK activation and inactivation rates, indicated by dashed lines. (B) Simulations with the newly developed upstream model (solid) provided excellent agreement with the model (P = 0.854, SSE = 0.038), much better than what was possible with the existing model structure (dashed) (P = 0.0002, SSE = 0.661). With the newly developed upstream and downstream modules integrated into the full model, new parameter estimates were able to fit both NF-κB (C) and IKK (D) activity in microglia. (E) Model predictions for the total amount of IκBα (solid line) are compared with experimental measurements of total IκBα protein in BV2-cells following 10 ng/ml TNFα treatment.