FIGURE 3.

NleB inhibits Lys⁶³-ubiquitination of TRAF3. A, cells were stimulated with 1.0 μg/ml poly(I:C) for 12 h. *, significantly different luciferase activity compared with HA control transfection (one-way ANOVA, n = 3). B, Raw 264.7 cells were transfected with TRAF3-FLAG and either treated with LPS (1.0 μg/ml, 30 min) or infected with C. rodentium strains for 3 h. Cells were lysed and immunoprecipitated using anti-FLAG antibody. Ubiquitination was assessed by immunoblotting for HA to detect ubiquitin (Ub) conjugates. C, quantification of Lys⁴⁸-ubiquitin from the experiments depicted in B. D, quantification of Lys⁶³-ubiquitin from the experiments depicted in B. *, significantly different ubiquitination levels compared with WT infection (one-way ANOVA, n = 3). E, Lys⁴⁸ and Lys⁶³ ubiquitination of TRAF3 as a function of NleB transfection. IP, immunoprecipitation; WB, Western blot.