Fig 2: Immunocapture of drug/TNF complexes by Type 3 antibodies. Type 3 anti-golimumab/TNF antibody AbD25705 and Type 3 anti-adalimumab/TNF antibody AbD20350 were coated on a microtiter plate at 1 µg/mL. Golimumab and adalimumab at a fixed concentration (1 µg/mL = 7 nM) were incubated for 1 hour with an increasing amount of TNF and added to the plate. Detection was performed with anti-human IgG:HRP antibody specific for the CH2 domain that detects golimumab or adalimumab but not the Fab antibodies, followed by QuantaBlu fluorogenic peroxidase substrate.

Fig 3: Comparison of assay formats. (a) Schematic view of the assay set-up: bridging assay set-up using a Type 1 reagent for capture and an HRP-conjugated Type 1 antibody for detection of a bivalent drug and (b) antigen capture assay using an HRP-conjugated Type 3 antibody for detecting a monovalent or bivalent drug in complex with its drug target. (c) Detection of adalimumab using a Type 3 antibody in antigen capture assay (black), or Type 1 capture and detection antibodies in a bridging assay (red). For the antigen capture assay, human TNF was coated at 5 µg/mL on a microtiter plate overnight. After washing and blocking, adalimumab spiked into 10% human serum was added. HRP-conjugated anti-adalimumab/TNF antibody AbD18754 (KD = 67 nM) was added at 2 µg/mL in HISPEC assay diluent, followed by QuantaBlu fluorogenic peroxidase substrate. For the adalimumab bridging assay, the anti-adalimumab Type 1 antibody AbD18654 (KD = 0.16 nM) was coated at 1 µg/mL on a microtiter plate overnight. After washing and blocking, adalimumab spiked into 10% human serum was added, followed by HRP-conjugated Type 1 anti-adalimumab antibody AbD18655 (KD = 0.06 nM) at 2 µg/mL in HISPEC assay diluent and QuantaBlu fluorogenic peroxidase substrate.

Fig 4: Synergistic toxicity of iron and inflammation is mediated by maternal TNFα.a Iron-loaded hepcidin KO dams were treated intravenously via the retroorbital sinus with 500 μg neutralizing TNFα antibody (nTNFα, white circles) or isotype control IgG (IgG, blue circles) neutralizing trinitrophenol 15 h prior to subcutaneous injection of LPS (0.5 μg/g) on E15.5 for 24 h. b Gross morphology and incidence of resorbing embryos. c–e Maternal liver expression of inflammatory markers Saa1, Il6, and Cxcl9 normalized to Hprt. f–i Placental mRNA expression of inflammatory markers Tnf, Il6, Il1b, and Cxcl9 normalized to Rpl4. j, k Western blots and quantitation of whole placenta or embryo liver lysates for cleaved caspase-3 expression normalized to β-actin. l TUNEL stain of placental sections. m Immunohistochemistry for cleaved caspase-3 (brown) and CD31 (red) in paraffin-embedded placenta, embryo heart, lung, and liver. l, m Representative images of n = 3 sections/group. Scale bar = 50 μm. f–m Embryo and placentas were randomly selected for analysis. c–k Error bars represent mean ± s.e.m. Statistical differences between groups were determined by two-tailed Mann–Whitney U or two-tailed Student’s t-test (denoted by *). P-values are indicated in each figure panel. Source data are provided as a Source data file.

Fig 5: The S513A mutation destabilizes Regnase-1 protein but does not affect target mRNA abundance.(A–C) Immunoblot analysis of Zc3h12aWT/WT and Zc3h12aS513A/S513A MEFs stimulated with IL-1β (10 ng/ml) (A), BMDMs stimulated with LPS (100 ng/ml) (B), and thioglycollate-elicited PECs stimulated with LPS (100 ng/ml) (C) for indicated time. PECs were pretreated with MG-132 (5 μM) 2 hr before the stimulation. (D)-(F) mRNA expression of Zc3h12a and Il6 in Zc3h12aWT/WT and Zc3h12aS513A/S513A MEFs stimulated with IL-1β (10 ng/ml) for 4 hr (D), BMDMs stimulated with LPS (100 ng/ml) for 4 hr (E), and thioglycollate-elicited PECs stimulated with LPS (100 ng/ml) for indicated time (F). (G)-(I) IL-6 secretion in Zc3h12aWT/WT and Zc3h12aS513A/S513A MEFs stimulated with IL-1β (10 ng/ml), IL-17A (50 ng/ml), or TNF (10 ng/ml) for 24 hr (G), BMDMs stimulated with Pam3CSK4 (1 or 10 ng/ml), poly I:C (10 or 100 μg/ml), LPS (10 or 100 ng/ml), R848 (10 or 100 nM), or CpG DNA (0.1 or 1 μM) for 24 hr (H), and thioglycollate-elicited PECs stimulated with LPS (100 ng/ml), R848 (100 nM), or IL-1β (10 ng/ml) for 24 hr (I). (J) Schematic representation of Model 1 in which 14-3-3-bound Regnase-1 does not have the function of degrading its target mRNAs. This model could explain the experimental observations. (K) Schematic representation of Model 2 in which 14-3-3-bound Regnase-1 maintains some ability to degrade its target mRNAs. This model is not consistent with the experimental observations. In (D)-(I), bars represent mean values of biological replicates (n = 3), and error bars represent standard deviation. Data is representative of two independent experiments, each with three biological replicates.