Figure 2.

Hoil-1l^{nzf∗/nzf∗} knockin mice display no developmental phenotype yet TNF-induced NF-κB activation is reduced in Hoil-1l^{nzf∗/nzf∗}cells

(A) Numbers of weaned and expected pups of the indicated genotypes from Hoil-1l+/nzf∗crosses.

(B) Gross appearance image of 11-week-old Hoil-1l^+/+ and Hoil-1l^{nzf∗/nzf∗} mice. Scale bar: 10 mm.

(C) H&E staining of the indicated tissues from 11-week-old Hoil-1l^+/+ and Hoil-1l^{nzf∗/nzf∗} mice. Scale bar: 200 μm.

(D) Immunoblotting to detect phosphorylation of IKK-α/β in Hoil-1l^+/+ and Hoil-1l^{nzf∗/nzf∗} MEFs treated with mouse TNF (20 ng/ml) for the indicated time. An α-Vinculin antibody was used to monitor loading amount. ∗ Indicates modified HOIL-1L.

(E) mRNA levels of NF-κB targets (TNF, IκB-α, A20, and ICAM) determined by qRT-PCR in Hoil-1l^+/+ and Hoil-1l^{nzf∗/nzf∗} BMDMs treated with mouse TNF (20 ng/mL) for the indicated time. Normalization was done to β-actin.

(F) Immunoblotting to detect TNFR Complex I formation in Hoil-1l^+/+ and Hoil-1l^{nzf∗/nzf∗} MEFs. Total cell lysates (Input) of cells treated with or without human TNF (100 ng/mL), and immunoprecipitates (IP: α-FLAG) were subjected to SDS-PAGE. Recruitment of HOIP, SHARPIN, and HOIL-1L was monitored by immunoblotting. An α-HOIL-1L antibody (Atlas Antibody, HPA 024185) was used for detecting HOIL-1L in TNFR Complex I and input. α-HOIL-1L (Merck Millipore, MABC576) was additionally used to detect HOIL-1L in input. α-Vinculin antibody was used to monitor loading amount. Data are representative of at least three independent experiments. (E) Data are represented as mean ± SD. ANOVA, n = 3, ∗∗p value ≤0.01, ∗∗∗p value ≤0.001, ∗∗∗∗p value ≤0.0001.