Figure 6.

OTULIN Deficiency Leads to Autoactivation of Macrophages

(A) Immunoblots of different polyUb chains in whole-cell lysate from untreated (i.e., no exogenous stimulation after differentiation) LysMCre-Otulin^flox BMDMs. (Right) Densitometry analysis of the Met1-Ub signal from above the 51 kDa marker in immunoblot experiments.

(B) Immunoblots of NF-κB signaling proteins from untreated LysMCre-Otulin^flox BMDMs.

(C) Immunoblot analysis of IκBα stability in LysMCre-Otulin^flox BMDMs treated with anti-TNF neutralizing antibodies or isotype control and cycloheximide (CHX) as indicated.

(D) Relative mRNA levels of Tnf, Nfkbia, Il6, and Tnfaip3 from untreated LysMCre-Otulin^flox BMDMs measured by quantitative RT-PCR. Each data point is mean of two technical replicas. Statistical significance was determined using two-tailed Student’s t test.

(E) Luminex analysis of TNF and IL-6 from cell culture supernatants of untreated LysMCre-Otulin^flox BMDMs. Cells were split, washed in PBS, and reseeded in fresh cell culture medium 24 hr prior to analysis. Results were pooled from two independent experiments.

(F) Viability of LysMCre-Otulin^flox BMDMs 10 hr after treatment. Each experiment was performed as biological duplicates. Results were normalized to LysMCre-Otulin^+/flox.

(G and H) Immunoblots of signaling proteins from untreated LysMCre-Otulin^flox BMDMs.

(I) Densitometry analysis of IκBα stability from experiments performed as in (C).

(J) Relative mRNA levels of Tnf measured by quantitative RT-PCR in LysMCre-Otulin^flox BMDMs treated with 1 ng/mL TNF as indicated (n = 3).

Data are presented as mean ± SEM, and n represents number of biological replicas. See also Figure S6.