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. Author manuscript; available in PMC: 2014 Jan 6.
Published in final edited form as: Inflamm Bowel Dis. 2012 Jan 23;18(5):10.1002/ibd.22883. doi: 10.1002/ibd.22883

Figure 3. Trib2 inhibits TLR5-mediated activation of NF-κB downstream of the TRAF6 complex.

Figure 3

(A) HEK293/TLR5 cells were transfected with increasing amounts of Trib2 or empty vector and NF-κB reporter with Renilla. Cells were stimulated with flagellin (100ng/ml, 6 hours) 24 hours post-transfection and luciferase assay was performed. Results are normalized to cells transfected with NF-κB in the absence of ligand stimulation (n=4. (B) Left panel: Quantitative RT-PCR of Trib2 was performed using RNA from HEK293/TLR5 cells after transfection with 20nM of Trib2 siRNA or control siRNA for 24 hours. Results are normalized to HEK293/TLR5 without siRNA (n=3). Right panel: HA-Trib2 (100ng/ml) was co-transfected with Trib2-specific or control siRNA (100nM) in HEK293/TLR5 cells. Lysates were collected 24 hours post-transfection and assessed by Western blot. (C) Upper panel: HEK293/TLR5 cells were transfected with Trib2 siRNA or control siRNA (20nM) or transfected with NF-κB reporter and Renilla only (Ctrl). Cells were stimulated with flagellin (100ng/ml, 6 hours) 24 hours post-transfection and luciferase assay was performed (n≥4). Results were normalized to cells transfected with NF-κB in absence of ligand stimulation. Lower panel: HEK293/TLR2, HEK293/TLR3 or HEK293/TLR5 cells were transfected with Trib2 siRNA or control siRNA (20nM). Cells were treated with Pam3CSK4 (1μg/ml, 6 hours), poly(I:C) (2.5μg/ml, 6 hours) or flagellin (100ng/ml, 6 hours) 24 hours post-transfection and luciferase reporter assay was performed (n≥3). Results were normalized to cells transfected with control siRNA. (D) Macrophages from Trib2 KO and WT mice were stimulated with flagellin (100ng/ml, 0 or 5 min) and cell lysates were collected. Membranes were first hybridized with phospho-IKKα/β or phospho-p65 antibodies, then stripped and rehybridized with anti- IKKα/β or anti-p65 antibodies. (E) MyD88 (200ng/ml) or TRAF6 (200ng/ml) were co-transfected with Trib2 (100ng/ml) or empty vector (100ng/ml) and NF-κB reporter with Renilla. Luciferase assay was performed 24 hours post-transfection. Results were normalized to cells transfected with NF-κB without MyD88 or TRAF6 (n≥4). (F) Western blots of the same cell lysates used for the luciferase assay were hybridized with MyD88 and TRAF6 antibodies. Membranes were stripped and rehybridized with anti-actin as the loading control. * p<0.05; ** p<0.01 for all panels.