Figure 2.

NLRC5 ubiquitination upon LPS treatment is critical for NLRC5–IKK-β interaction dynamics. (A–C) Cell lysates of HEK293T/TLR4 cells (A), RAW264.7 cells (B), or MEFs (C) were collected at the indicated time after LPS (200 ng/ml) treatment and immunoprecipitated with anti-NLRC5 antibody, followed by immunoblotting with the indicated antibodies. (D) Quantification of A–C. IKK-β–NLRC5 complex (red) and ubiquitinated NLRC5 (blue) in HEK293T/TLR4, RAW264.7, and MEFs. (E) Proposed model II incorporating NLRC5 ubiquitination (shaded square). (F) Model fits to experimental data measured in our previous work (Cui et al., 2010). Blue curves indicate the Model II simulation. Red circles indicate experimental data. (G) Local peak sensitivities for all kinetic parameters and nonzero initial conditions in Model II for total pIKK-β (blue bars) and ubiquitinated NLRC5 (ubNLRC5, red bars). Φ, null; χ², the sum of deviations; IB, immunoblotting; IP, immunoprecipitation; N, number of experimental points; WCL, whole cell lysate.