Interaction of p65 and p50 with eGFP-NleC1–237 and critical role for NleC residues 33–64. A, representative immunoblot of anti-GFP immunoprecipitate isolated from cells transfected with pEGFP, pEGFP-NleC, or pEGFP-NleC1–237 probed for p65 and p50. The data reveals that p65 and p50 can be isolated with eGFP-NleC1–237 but not eGFP or eGFP-NleC, the latter presumably due to rapid p65/p50 degradation. B, schematic of NleC N-terminal truncation variants constructed and screened in NFκB luciferase reporter assay. C, relative luciferase activity of cells expressing NleC and indicated variants, compared with control pEGFP-transfected cells. Data shown are mean (±S.D.) of three experiments done in triplicate with level of significance (Student's t test) indicated. *, p ≤ 0.05; ***, p ≤ 0.005 as compared with empty vector control. NleC33–330 but not NleC65–330 inhibits NFκB luciferase reporter activity revealing a critical role for residues between 33 and 64. D, representative immunoblot probed for GFP, p65, and actin that links loss of NFκB luciferase reporter activity of the NleC65–330 variants with a major defect in depleting p65 from the cell.