Fig. 3

B. melitensis UGPase inhibits the activation of NF-κB by modulating the ubiquitination of NEMO. Cells were infected with 16 M and 16 M-UGPase⁻ for 6 or 11 h. Mock-infected cells were used as negative controls. Whole-cell extracts were collected and divided into two parts. One part was used for coimmunoprecipitation, and the other part was used for immunoblotting analysis. β-actin was used as a negative control for the immunoblotting analysis. (A-B) Results of Co-IP at 6 hpi. K63-Ub of NEMO (A) and Met1-Ub of NEMO (B). Left panel, total ubiquitinated K63 or Met1; right panel, ubiquitinated K63 or Met1 of NEMO. (C-D) Results from the Co-IP at 11 hpi. K63-Ub of NEMO (C) and Met1-Ub of NEMO (D). Left panel, total ubiquitinated K63 or Met1; right panel, ubiquitinated K63 or Met1 of NEMO. (E-F) Expression levels of IκBα and p-P65 at 6 hpi (E) and 11 hpi (F). (G-H) Levels of the regulated NF-κB at 6 (G) and 11 hpi (H). The activation of the NF-κB signaling pathway was evaluated using a Dual-Luciferase® Reporter 1000 assay system. Unprocessed original scans of the Western blots can be found in Supplementary Fig. S1, S2, S3, S4, S5, S6