ril 27  (R&D Systems)

Journal: Science advances

Article Title: Human interleukin-12α and EBI3 are cytokines with anti-inflammatory functions.

doi: 10.1126/sciadv.adg6874

Figure Lengend Snippet: Fig. 1. IL-12α and EBI3 mutually promote their secretion and form IL-35. (A) IL- 35 (alphaFold2 docked model) shares its α subunit IL-12α with IL-12 [Protein Data Bank (PDB): 3HMX], and its β subunit EBI3 with IL-27 (PDB: 7u7n). To assess mutu- ally induced secretion of IL-12α and EBI3, constant DNA amounts of IL-12α were cotransfected with increasing DNA amounts of EBI3 (B) or vice versa (C). Both IL- 12α and EBI3 show a reduced mobility in the medium, indicating that both sub- units are retained in the endoplasmic reticulum in isolation and traverse the Golgi during secretion, as indicated by modification of their glycans. (D) Co-IP of FLAG- tagged IL-12α coexpressed with EBI3 in the cell medium verifies assembly for these two proteins. Quantification of the IL-12α IP efficiency and the fraction of EBI3 that is found in complex with IL-12α is shown (n = 3 ± SD). (E) Co-IP of secreted HA- tagged EBI3 coexpressed with IL-12αFLAG verifies assembly for these two proteins in the medium. Quantification of the EBI3HA IP efficiency and the fraction of IL- 12αFLAG that is found in complex with EBI3HA is shown (n = 5 ± SD). Constructs were expressed in human embryonic kidney (HEK) 293T cells. One representative immunoblot is shown in each case.

Article Snippet: For co-IP experiments with purified proteins, 1 μg of IL-12αC96S,His and an equimolar amount of IL-12βC199S (previously purified in our laboratory), 1 μg of EBI3 and equimolar mIL-27α (R&D Systems, 7430-ML-010), or 1 μg of IL-12αC96S,His and equimolar EBI3 were mixed to a final volume of 200 μl (PBS) and incubated for 1 hour at room temperature.

Techniques: Isolation, Modification, Co-Immunoprecipitation Assay, Construct, Western Blot

Journal: Science advances

Article Title: Human interleukin-12α and EBI3 are cytokines with anti-inflammatory functions.

doi: 10.1126/sciadv.adg6874

Figure Lengend Snippet: Fig. 2. IL-35 subunits can be secreted as nonheterodimers in contrast to IL-12 and IL-27. (A) EBI3 induces the secretion of IL-12α even when it is retained in the ER (via a C-terminal KDEL sequence). EBI3-induced secretion is observed for wild-type IL- 12α and a variant lacking the cysteine that forms an interchain disulfide bond in IL-12 (C96S). (B) The same as in (A), only that IL-12α was furnished with a KDEL ER-retention sequence and secretion of EBI3 was monitored. EBI3 secretion was slightly increased by coexpression with both IL-12α and the C96S variant. Quantifications of EBI3 secretion are shown below the blot. (C) A similar analysis for IL-12 reveals that IL-12βC199S, with an ER retention sequence, does not induce secretion of free IL- 12α but instead co-retains it in the cell. The same is observed for the combination of IL-12αC96S with IL-12βC199S, with both pro- teins lacking the cysteines that form the interchain disulfide bond in IL-12. (D) Coexpression of wild-type EBI3 and IL-27α leads to the secretion of IL-27. When IL-27α was ER-retained (IL- 27αKDEL), EBI3 secretion was reduced, in contrast to (B). (E) Free IL-12α can be detected in medium samples after pulldown of EBI3. IL-12α and EBI3 are cotransfected, and cell supernatants underwent two consecutive HA-IPs to isolate EBI3HA-containing complexes. IL-12α is also coimmunoprecipitated as can be seen in the IL-12α blot after HA-IP I and II, indicating pulldown of IL- 35. The final IL-12α–IP reveals remaining IL-12α in the medium that is not interacting with EBI3. Band intensities show a sig- nificantly higher amount of free IL-12α when coexpressed with EBI3 (or EBI3KDEL) compared to transfection in isolation without EBI3.

Article Snippet: For co-IP experiments with purified proteins, 1 μg of IL-12αC96S,His and an equimolar amount of IL-12βC199S (previously purified in our laboratory), 1 μg of EBI3 and equimolar mIL-27α (R&D Systems, 7430-ML-010), or 1 μg of IL-12αC96S,His and equimolar EBI3 were mixed to a final volume of 200 μl (PBS) and incubated for 1 hour at room temperature.

Techniques: Sequencing, Variant Assay, Transfection, Isolation

Journal: Science advances

Article Title: Human interleukin-12α and EBI3 are cytokines with anti-inflammatory functions.

doi: 10.1126/sciadv.adg6874

Figure Lengend Snippet: Fig. 3. Recombinant human IL-12αC96S and EBI3 are stable and well-structured proteins. (A) Analysis of purified IL-12αC96S and EBI3 by reducing and nonreducing SDS–polyacrylamide gel electrophoresis (SDS-PAGE). Faster migration on nonreducing SDS-PAGE indicates the presence of disulfide bonds. Positions of intramolecular disulfide bonds are indicated in each subunit structure. (B) Reconstituted IL-12 and IL-27 are able to induce receptor heterodimerization. COS-7 cells were cotransfected with the indicated receptor chains equipped with the NanoBRET reporter system. Cells were stimulated with purified IL-12αC96S, which was previously incubated with recombinant human IL-12βC199S, or EBI3, previously incubated with murine IL-27α or the isolated subunits as indicated (10 nM final concentrations). Graphs represent the normalized NanoBRET signal (n = 3 ± SD). Statistical significance was calculated by one way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test; ****P < 0.0001 compared with the corresponding phosphate-buffered saline (PBS) control. (C) NK-92 or BL-2 cells were stimulated with preincubated IL-12αC96S + IL- 12βC199S or EBI3 + mIL-27α or the heterodimeric cytokines (10 ng/ml final concentrations). Downstream signaling was detected by STAT-phosphorylation via immuno- blot. (D) Far-UV CD spectra for IL-12αC96S and EBI3. (E) IL-12αC96S unfolds cooperatively with an apparent melting temperature of 47 ± 0.2°C and EBI3 with an apparent melting temperature of 50 ± 0.2°C (green line, experimental data; black line, Boltzmann sigmoidal nonlinear curve fit; transitions were not reversible).

Article Snippet: For co-IP experiments with purified proteins, 1 μg of IL-12αC96S,His and an equimolar amount of IL-12βC199S (previously purified in our laboratory), 1 μg of EBI3 and equimolar mIL-27α (R&D Systems, 7430-ML-010), or 1 μg of IL-12αC96S,His and equimolar EBI3 were mixed to a final volume of 200 μl (PBS) and incubated for 1 hour at room temperature.

Techniques: Recombinant, Purification, Polyacrylamide Gel Electrophoresis, SDS Page, Migration, Incubation, Isolation, Saline, Control, Phospho-proteomics, Circular Dichroism

Journal: Science advances

Article Title: Human interleukin-12α and EBI3 are cytokines with anti-inflammatory functions.

doi: 10.1126/sciadv.adg6874

Figure Lengend Snippet: Fig. 5. IL-12αC96S and EBI3 show different sig- naling characteristics. (A) The NanoBRET re- porter system indicates IL-12 receptor heterodimerization after stimulation with 1 nM IL- 12, which can be blocked by a 30-min pretreat- ment with IL-12βC199S (1, 10, or 100 nM). (B) In the same assay, a 30-min pretreatment with IL- 12αC96S (1, 10, or 100 nM) shows reduced IL-12– induced receptor heterodimerization at a 10-fold excess of IL-12αC96S. (C) A preincubation with in- creasing concentration of EBI3 did not have any effect on IL-27 activity. Graphs represent the nor- malized NanoBRET signal (n = 3 ± SD). (D) STAT4 phosphorylation in NK-92 cells after treatment with IL-12 (1 ng/ml) is blocked by a 30-min pre- treatment with IL-12βC199S (10 and 100 ng/ml) or IL-12αC96S (10 ng/ml). Representative immuno- blots from one of three independent experiments are shown. (E) BL-2 cells were used to monitor IL- 27 (10 ng/ml)–mediated STAT1 phosphorylation, which is not blocked by pretreatment with EBI3 (10, 100, or 1000 ng/ml) for 30 min (n = 3 ± SD). (F) NK-92 or (G) BL-2 cells were pretreated with IL- 12αC96S (10 ng/ml) or EBI3 (10 ng/ml) overnight (o/n) before addition of IL-12 or IL-27, respec- tively, to investigate receptor internalization. (n = 3 ± SD, *P < 0.05 and ****P < 0.0001) (H) STAT1 phosphorylation in human PBMCs treated with EBI3 and IL-12αC96S compared to the cytokines IL- 10, IL-27, and IL-35 and their effect after HDM stimulation. Representative immunoblots from one of three independent experiments are shown.

Article Snippet: For co-IP experiments with purified proteins, 1 μg of IL-12αC96S,His and an equimolar amount of IL-12βC199S (previously purified in our laboratory), 1 μg of EBI3 and equimolar mIL-27α (R&D Systems, 7430-ML-010), or 1 μg of IL-12αC96S,His and equimolar EBI3 were mixed to a final volume of 200 μl (PBS) and incubated for 1 hour at room temperature.

Techniques: Concentration Assay, Activity Assay, Phospho-proteomics, Western Blot