Figure 6.

OTULIN Overexpression Inhibits TNFα Signaling

(A) Luciferase assays performed as in Figure 4B in HEK 293ET cells transfected with OTULIN, A20, or both OTULIN and A20 and stimulated with TNFα (100 ng/ml) or poly(I:C) (10 μg/ml). Western blots below show transfected protein levels. Error bars represent SD from the mean for experiments performed in triplicate. p values are given to indicate significance. ^*, the control set to 1.

(B–F) Stable doxycycline-inducible control and OTULIN-overexpressing T-REx 293 cells after treatment with 1 μg/ml doxycyclin (Dox) for 24 hr (see the Extended Experimental Procedures).

(B) Quantitative PCR (qPCR) analysis of selected NF-κB target genes in control (gray) and OTULIN-overexpressing cells (black) after treatment with 10 ng/ml TNFα for the indicated times.

(C) Analysis of TNFα-stimulated NF-κB activation and IκBα phosphorylation upon TNFα treatment (100 ng/ml) over the indicated time course by western blotting with indicated antibodies and EMSA. See (F) for tubulin control.

(D) Clonogenic survival of indicated cell lines ± Dox and ± TNFα (50 ng/ml) for 24 hr (see the Extended Experimental Procedures).

(E) Cell viability counts of cells treated as in (D). Error bars represent SD from the mean for experiments performed in triplicate.

(F) Analysis of TNFα-stimulated signaling cascades upon TNFα treatment (100 ng/ml) over the indicated time course by western blotting.