Fig 2: ABIN-2–A20 interaction negatively regulates HDM-induced allergic responses. (A) Schematic representation of ABIN-2 domains and protein interactions. (B) FLAG-A20 was transiently expressed in HEK293 cells. Interaction with GST-ABIN-2 and GST-ABIN-2E256K was determined in pulldown assays (left). A20 binding was quantified from three independent experiments. Unpaired two-tailed t test. EV, empty vector; WCL, whole cell lysate. (C) Lysates of BMDCs generated from the indicated genotypes were probed for ABIN-2, TPL-2, and NF-?B1 p105. Data are representative of three independent experiments. (D) Lysates from LPS-stimulated BMDCs were immunoblotted for the indicated antigens. Data are representative of two independent experiments. (E) Schematic representation of HDM/Alum-dependent allergy model (Model 1). o.p., oropharyngeal. (F) Differential cell counts in BAL fluids of PBS and HDM-challenged WT, Map3k8D270A/D270A, and Tnip2E256K/E256K mice. (G) Cytokines mRNA expression levels in the lung, as assessed by qRT-PCR. (H) Total IgE levels in blood serum, as assessed by ELISA. (I) H&E-stained lung sections (left) and inflammation scores (right) from PBS and HDM-challenged mice. Data in panels F–I are shown as mean ± SEM and are pooled from three independent experiments (n = 10–15 mice/genotype). *, P < 0.05; **, P < 0.005; ***, P < 0.001. Comparisons assessed by Kruskal-Wallis and Dunn-Bonferroni’s post hoc test. ns, not significant.

Fig 3: Toxic APAP exposure induces proinflammatory NF?B signaling in the distal lung. (a) Representative Western blot and densitometric analysis of NF?? inhibitory proteins p105, I?Ba, and I?Bß in distal lung cytosolic extracts after APAP exposure (2, 4, and 8 hours; 280 mg/kg, IP). GAPDH is shown as loading control. Data is expressed as mean ± SEM (N = 4-6 per time point). *p < 0.05 vs. unexposed control. (b) Representative Western blot and densitometric analysis of NF?? subunits p65 and p50 in distal lung nuclear extracts after APAP exposure (2, 4, and 8 hours; 280 mg/kg, IP). HDAC is shown as a loading control. GAPDH is shown to confirm the purity of the nuclear sample. Data is expressed as mean ± SEM (N = 4-6 per time point). *p < 0.05 vs. unexposed control. (c) Fold change in NF?? target gene mRNA expression: Mmp9, Ccl2, Il1b, Nfkbia, Gclc, and Ptgs2 following APAP exposure (8 hours; 280 mg/kg, IP). Data expressed as mean ± SEM (N = 4-6 per time point). *p < 0.05 5 vs. unexposed control.

Fig 4: Noncanonical NF-KB is required for the apoptosis resistance in PELI1-knockdown cells. (A–D) Flow cytometry analysis of the apoptosis frequencies of control, PELI1-knockdown, NIK-knockdown or PELI1/NIK double knockdown TE-1 and ECA-109 cancer cells left non-treated (NT) or treated with 10 Gy IR (IR). The data are presented as representative plots (A, B) and a summary bar graph (C, D) (n = 4). (E, F) Immunoblot analysis of NF-?B, ACTIN, and lamin B (loading controls) in cytoplasmic extracts (CE) and nuclear extracts (NE) (E), and of the of cleaved PARP, caspase 3, caspase 9, caspase 7, Bcl-XL, Bax, and ACTIN (loading controls) in whole cell lysates (F) of control, PELI1-knockdown, NIK-knockdown or PELI1/NIK double knockdown in TE-1 cancer cells left untreated (-) or treated with 10 Gy IR (+). (G) Proposed model illustrating the mechanism of PELI1 regulating the sensitivity of radiotherapy through modulating noncanonical NF-?B. Each panel is representative of at least three independent experiments. Data with error bars represent mean ± SEM. *P < 0.05 as determined by two-way ANOVA with a Bonferroni post-test (C, D).