(A–B) HEK293 cells stably transfected with Trib2 were stimulated with flagellin. Eluents from FLAG and Strep II beads were separated by SDS-PAGE and visualized by (A) colloidal Coomassie and (B) immunoblotting with FLAG antibody. (C) HEK293/TLR5 cells were transfected with FLAG-Trib2 or FLAG vector for 24 hours followed by stimulation with flagellin for 0 or 20 minutes. Lysates were immunoprecipitated with FLAG antibody and blotted for NF-κB2. (D) Same lysate as (C), immunoprecipitated with NF-κB2 antibody and blotted for FLAG. (E) MyD88 (200ng/ml) was co-transfected in HEK293/TLR5 cells with Trib2 (100ng/ml), NF-κB2 (500ng/ml) or empty vector and NF-κB reporter with Renilla. Luciferase assay was performed 24 hours post-transfection. Results were normalized to cells transfected with NF-κB but no MyD88 (n≥4). #, difference between Trib2 alone and Trib2 with NK-κB2, p<0.05. (F) Upper panel: HEK293/TLR5 cells were transfected with FLAG-Trib2 or FLAG vector for 24 hours followed by stimulation with flagellin for 0 or 5 minutes. Lower panel: HEK293/TLR5 cells were transfected with siCtrl or siTrib2 for 24 hours, followed by stimulation with flagellin for 0 or 5 minutes. Membranes were first hybridized with phospho-NF-κB2, then stripped and rehybridized with anti-NF-κB2 antibody. * p<0.05 for all panels.