FIG 5 .

TLR4 stimulation induces HOIP conformational change. (A) Diagram of HOIP-FRET constructs with amino-terminal YFP fusions and carboxyl-terminal CFP fusions. (B) Function of HOIP-FRET constructs as determined by immunoblot for linear ubiquitins in 293T cell lysates containing either HOIP-WT or HOIP-FRET. (C) HOIP-FRET constructs were transfected into 293T cells. At 24 h after transfection, cells were lysed and FRET was measured. FRET values were calculated by subtracting background fluorescence and dividing fluorescence of YFP by fluorescence of CFP. Data are representative of three independent experiments. (D) HOIP-FRET constructs were transfected into A20.2J HOIP^−/− cells by using the Neon system (Life Technologies). At 24 h after transfection, cells were resuspended in FluoroBrite medium, and fluorescence was measured using an Envision plate reader. After addition of LPS, fluorescence readings were taken at the indicated times. Relative FRET was calculated as FRET(x min)/FRET(0 min). HOIP-eCFP and HOIP-eYFP constructs were used as negative controls. Data are representative of three independent experiments.