PMC

(A) Flow cytometry with CD3 and CD19 antibodies shows that whereas CD19⁻CD3⁺ cells (T cells) are similar in number, C19⁺CD3⁻(B cells) are greatly diminished in the X^WTX^NemoKi mice.
(B) Graphical representation of the fluorescent intensity of CD19 and CD3 staining, again showing diminished B cells in the X^WTX^NemoKi mice.
(C) Absolute number of splenic B and T cells in the WT and X^WTX^NemoKi mice. Similar results were found in a total of three mice from each genotype.

(A) C57BL/6 TNFR1^−/− mice were mated with TNFR1^+/+X^WTX^NemoKi mice and the resulting TNFR1^+/−X^WTX^Nemoki mice were mated with TNFR1^−/−X^WTY mice to obtain TNFR1^−/−X^WTX^NemoKi mice, which were further mated with TNFR1^−/−X^WTY to generate TNFR1^−/−X^NemoKiY mice. The Mendelian ratios of this mating are shown. In a WT TNFR1 background, we did not obtain pure NEMO knockin mice; however, when TNFR1 was deleted, we obtained male nonubiquitinatable NEMO knockin mice, suggesting that TNFR1 deletion complements the nonubiquitinatable NEMO defect.
(B) Kaplan-Maier curves showing significantly decreased lifespans in the TNFR1^−/−X^NemoKiY and TNFR1^−/−X^NemoKiX^NemoKi mice.
(C) Histologic evidence of steatohepatitis in the TNFR1^−/−X^NemoKiY mice, including macro- and microvesicular steatosis, inflammatory infiltrates most prominent in zone 3 of the liver, and prominent nucleolar vacuolization (lower panel, nucleolar vacuolization is shown by arrows). These are all features of NASH.
(D) Elevated liver function tests in the TNFR1^−/−X^NemoKiY mice, indicating significant hepatocyte injury. SEM, *p < 0.05, **p < 0.01, ***p < 0.001.

(A) Primary BMDMs were generated from TNFR1^−/−X^WTY, TNFR1^−/−X^NemoKiY, or TNFR1^+/+X^WTY mice and were treated with 10 μg/ml of MDP for the indicated time period. BMDMs containing nonubiquitinatable NEMO showed an inability to phosphorylate two IKK substrates, IκBα and p105, in response to MDP. The inability to phosphorylate p105 further leads to an inability to activate p44/p42 ERK in response to MDP.
(B) Primary BMDMs were generated from TNFR1^−/−X^WTY or TNFR1^−/−X^NemoKiY mice and were treated with 10 μg/ml of MDP for the indicated time period. Lysates were generated in denaturing conditions (boiled with 1% SDS). After cooling, the SDS was diluted to 0.1% and NEMO immunoprecipitation was performed. Western blotting showed that although WT NEMO could be inducibly ubiquitinated, knockin NEMO could not.
(C) To quantitate the signaling effect and to determine the role of NEMO ubiquitination in MDP-induced gene expression, the indicated BMDMs were treated with 10 mg/ml MDP for 4 hr before RNA was harvested and subjected to qRT-PCR. In all cases studied, the BMDMs expressing nonubiquitinatable NEMO showed greatly decreased MDP-induced gene expression. SEM, *p < 0.05, **p < 0.01, ***p < 0.001.

(A) Gross photograph of a representative 8-month-old nonubiquitinatable NEMO heterozygous female compared with a WT mouse. The nonubiquitinatable NEMO heterozygous mice develop ulcerated and plaque-like lesions distributed throughout the body. Histologically, the plaque-like skin lesions show hyper-keratosis, parakeratosis, keratin pearls, and a hyperproliferative epidermis with rete-ridge-like protrusions into the dermis. Nonlesional skin from the nonubiquitinatable heterozygous NEMO knockin mice shows little histological change relative to WT mice.
(B) qRT-PCR analysis of cytokines from the skin of WT littermates or from lesional or nonlesional skin from heterozygote nonubiquitinatable NEMO knockin mice (n = 3). Cytokines are significantly elevated in both the lesional and nonlesional skin from the nonubiquitinatable NEMO heterozygous mice. SEM, *p < 0.05, ***p < 0.001.
(C) Representative gross picture showing the enlarged spleens present in the heterozygous nonubiquitinatable NEMO female mice relative to WT female littermate control mice. Flow-cytometry gating on the different subsets of splenic immune cells reveals that the percentage of Gr₁⁺CD11_b⁺ cells is greatly increased, suggesting an inflammatory phenotype in the heterozygous nonubiquitinatable NEMO mice.

(A) Primary BMDMs were generated from TNFR1^−/−X^WTY, TNFR1^−/−X^NemoKiY, or TNFR1^+/+X^WTY mice and were treated with 25 ng/ml of LPS for the indicated time period. Although the initial levels of total IκBα dropped in all three genetic lines, only BMDMs with WT NEMO could sustain an NF-κB response, as indicated by phospho-IκBα and phospho-p105 blots. Erk signaling was likewise substantially diminished.
(B) To quantify and verify these signaling results, the indicated BMDMs were treated with LPS (3 ng/ml) for 4 hr. RNA was harvested and subjected to qRT-PCR. Although all three genetic lines showed an increase in KC or TNF-α production, this was severely decreased in the BMDMs from TNFR1^−/−X^NemoKiY mice. SEM, *p < 0.05, **p < 0.01.
(C) The indicated BMDMs were treated with IL-1 (5 ng/ml) for 4 hr before cells were harvested and subjected to qRT-PCR. IL-1-induced KC and IL-6 expression was significantly impaired in the TNFR1^−/−X^NemoKiY BMDMs. SEM, *p < 0.05.
(D) The indicated BMDMs were treated with IFN-γ (2 ng/ml) for 4 hr before cells were harvested and subjected to qRT-PCR. There were no significant changes in gene expression between any of the cell lines. SEM.

(A) Schematic of the targeting construct used to generate a nonubiquitinatable NEMO mouse. Exons 2–5 were replaced with a cDNA encoding NEMO in which lysines 285 and 399 were replaced with arginines such that ubiquitination could not occur. The construct was tagged on the N terminus with a myc tag such that it could be differentiated from endogenous NEMO. Additionally, the NEO cassette was flanked by FRT sites such that when it was generated, the mouse could be mated with a Flippase mouse to remove the NEO cassette.
(B) Stably transfected ES clones were assayed for nonubiquitinatable NEMO expression. An anti-myc western blot showed expression of the nonubiquitinatable myc gene, and an anti-NEMO blot showed relative expression of endogenous knocked-in NEMO in the ESCs (upper panels). The lower panel shows germline transmission in heterozygous female mice.
(C) Mendelian ratios of matings of WT male BL/6 mice with heterozygous female NEMO knockin mice. NEMO is on the X chromosome, and no NEMO knockin males were observed in the >200 mice generated.