Figure 4. TRIB proteins compete with each other for MAPKK binding.

(A) The relative intensity of the MKK4-TRIB PCA complexes were analysed in the presence of an increasing dose of unlabelled tribbles. N ≥ 3 (▼: TRIB1, ■: TRIB2, ▲: TRIB3) (B). Left panel: 400 ng expression plasmids, encoding for individual tribbles-V2 fusion proteins were transfected into HeLa cells and expression levels were detected by an anti-GFP western blot. Middle and right panels: Tribbles expression levels in HeLa cells increase in a dose dependent manner. Transfected doses of untagged tribbles, relative to the TRIB-PCA dose used in panel A are indicated above the individual panels. Representative western blots are shown (N = 3). (C) The differential impact of knockdown of specific tribbles on p38 MAPK activation was assessed in THP-1 cells. Cells transfected with non-targeting control or si-Trib constructs were stimulated by LPS for the stated length of time and the activation of p38 was detected by a phospho-p38 specific western blot. As loading control, the membrane was re-probed for β-actin. Upper panel: a representative result, Lower panel: quantitative assessment of p-p38 from three independent experiments. Data is expressed relative to the β-actin signal.