Figure 1.

UBE2L3 regulates cytokine-driven NF-κB activation. (A) NF-κB firefly luciferase reporter was cotransfected into HEK293 cells with control Renilla luciferase and 10 LUBAC HCIPs or UBE2D3. Luciferase activity was measured after 48 h. Data are pooled from 3 experiments. Standard deviation bars are shown. ^*P < 0.001. Western blot shows expression levels from one representative experiment. (B) HEK293 cells were treated with 10 ng/ml TNFα for the indicated times before cell lysis. Immunoprecipitation with anti-HOIL-1L antibody was followed by probing blot with the indicated antibldies. Whole cell lysates (WCL) were used as controls to compare input levels. (C) HEK293 cells were treated with control or UBE2L3 siRNA, after 48 h cells were stimulated with 10 ng/ml TNFα for the indicated period. Cell lysates were immunoprecipitated with anti-NEMO antibody and blotted as indicated. (D) HEK293 cells were transfected with scrambled siRNA control, UBE2L3 siRNA or UBE2D3 siRNA. After 48 h the cells were stimulated with 10 ng/ml TNFα for 2 or 4 h. Relative mRNA levels for the indicated NF-κB-dependent genes were determined by qPCR. Standard deviation bars are depicted. ^*P < 0.001. (E) After transfection with si-UBE2L3, si-control or si-UBE2L3 plus a rescue UBE2L3 cDNA, HeLa cells were stimulated with 10 ng/ml TNFα for 0 or 5 min. Cells were stained for detection of p65 and nuclei (DAPI). (F) HEK293 cells were transfected with siRNA control, UBE2L3 siRNA, or UBE2D3 siRNA. After 48 h the cells were stimulated with 10 ng/ml TNFα for the indicated times. Binding of nuclear p65 to consensus NF-κB DNA elements was assayed with anti-p65 antibody, developed with a secondary HRP-conjugated anti-IgG antibody and measured by luminescence. Standard deviation bars are depicted. ^*P < 0.001. (G) HEK293 cells were treated with si-control or si-UBE2L3. After 48 h cells were stimulated with TNFα for the indicated times. Cell lysates were immunoprecipitated with anti-TNFR1antibodies and probed with the indicated antibodies.