hek blue il 18 sensor cells  (InvivoGen)

Journal: mAbs

Article Title: Structures of clinical antibodies bound to IL-33 uncover two distinct epitopes underlying differential efficacy

doi: 10.1080/19420862.2026.2639673

Figure Lengend Snippet: Characterization of the three anti-IL-33 antibodies. (A) Sequence alignment of etokimab, itepekimab, and tozorakimab. CDRs and FRs were defined according to the IMGT system. (B) SPR analysis measuring the binding affinity of the three antibodies and ST2 to IL-33. Serially diluted IL-33 (starting concentration 11.11 nM) was injected over immobilized ligands for a 180 s association and 300 s dissociation. (C) Inhibition of IL-33-induced signaling by the antibodies. HEK-Blue IL-33 reporter cells were stimulated with IL-33 at 2 ng/mL (triangles) or 30 ng/mL (circles) in the presence of a series of each antibody. After 22 hours, SEAP activity in the supernatant was quantified by measuring absorbance at 620 nm. Itepekimab, etokimab, and tozorakimab are shown in blue, green, and purple, respectively. Data points represent mean ± SD ( n = 3). Dose–response curves were fitted, and IC 50 /IC 90 values were calculated using GraphPad Prism which were summarized in the right panel table. (D) Blocking of IL-33–induced signaling on human peripheral blood mononuclear cell (PBMCs). Antibody and 1 nM IL-33 were added to human PBMCs for 19 hours. IFN-γ release was measured by means of ELISA. All data are represented as means ± SDs.

Article Snippet: HEK-BlueTM IL-33 cells (InvivoGen, catalog no. hkb-hil33) were utilized for bioactivity assessment of IL-33 through monitoring NF-κB and AP-1 pathway activation.

Techniques: Sequencing, Binding Assay, Concentration Assay, Injection, Inhibition, Activity Assay, Blocking Assay, Enzyme-linked Immunosorbent Assay

Journal: mAbs

Article Title: Structures of clinical antibodies bound to IL-33 uncover two distinct epitopes underlying differential efficacy

doi: 10.1080/19420862.2026.2639673

Figure Lengend Snippet: Structural overview of antibody – IL-33 complexes. (A) Structures of IL-33 (medium orchid) in complex with etokimab Fab (salmon), itepekimab Fab (olive drab), and tozorakimab Fab (dark turquoise), shown from left to right. The rightmost panel shows a superposition of all three Fab – IL-33 complexes, illustrating their relative positions and orientations. (B) Structure of the IL-33–ST2 complex (dark slate blue; PDB: 4KC3). The interface of D1/D2 domains and IL-33 named Site 1, interface of D3 domain and IL-33 named Site 2. (C) Structural alignment of the three Fab – IL-33 complexes with the ST2–IL-33 complex, showing the spatial relationship between each antibody and ST2. Colors are consistent with panels (A) and (B).

Article Snippet: HEK-BlueTM IL-33 cells (InvivoGen, catalog no. hkb-hil33) were utilized for bioactivity assessment of IL-33 through monitoring NF-κB and AP-1 pathway activation.

Techniques:

Journal: mAbs

Article Title: Structures of clinical antibodies bound to IL-33 uncover two distinct epitopes underlying differential efficacy

doi: 10.1080/19420862.2026.2639673

Figure Lengend Snippet: Interactions of etokimab and itepekimab with IL-33. (A, B) Epitopes on IL-33 (medium orchid) and paratopes on (A) etokimab (heavy chain, salmon; light chain, orange) and (B) itepekimab (heavy chain, olive drab; light chain, coral) are colored in dark gray, binding residues within are highlighted. (C, D) Detailed interaction networks for (C) Etokimab – IL-33 and (D) Itepekimab – IL-33 complexes. Hydrogen bonds and salt bridges are indicated by black dashed and solid lines, respectively; a red solid line denotes an interaction involving both. Interaction residues within are highlighted in related color. (E) Structural footprint of etokimab (salmon), itepekimab (olive drab), tozorakimab (dark turquoise), and ST2 (dark slate blue) on IL-33 (dark gray). Shared binding residues are listed in the lower panel.

Article Snippet: HEK-BlueTM IL-33 cells (InvivoGen, catalog no. hkb-hil33) were utilized for bioactivity assessment of IL-33 through monitoring NF-κB and AP-1 pathway activation.

Techniques: Binding Assay

Journal: mAbs

Article Title: Structures of clinical antibodies bound to IL-33 uncover two distinct epitopes underlying differential efficacy

doi: 10.1080/19420862.2026.2639673

Figure Lengend Snippet: Molecular basis of tozorakimab recognition of IL-33. (A) Binding interface between tozorakimab and IL-33. The epitope on IL-33 is shown in dark gray, with key contacting residues highlighted. The paratope is composed of the heavy chain (dark turquoise) and light chain (yellow-green). (B) Structural footprint comparison between tozorakimab and ST2 on IL-33. The tozorakimab footprint is shown in dark turquoise, while the ST2 Site 1 interface is colored dark slate blue. Shared binding residues are colored and highlighted according to their chain origin in tozorakimab. (C) Detailed molecular interactions in the tozorakimab – IL-33 complex. Hydrogen bonds and salt bridges are indicated by black dashed and solid lines, respectively; a red solid line denotes an interaction involving both. Relevant residues are colored consistently with panels A and B. (D) Electrostatic surface potential analysis of IL-33 (left) and tozorakimab (right). Surfaces are colored from red (negative charge) to blue (positive charge). The binding footprints of tozorakimab (dark turquoise) and IL-33 (medium orchid) are outlined. Dashed circles indicate complementary charged patches that facilitate binding.

Article Snippet: HEK-BlueTM IL-33 cells (InvivoGen, catalog no. hkb-hil33) were utilized for bioactivity assessment of IL-33 through monitoring NF-κB and AP-1 pathway activation.

Techniques: Binding Assay, Comparison

Journal: mAbs

Article Title: Structures of clinical antibodies bound to IL-33 uncover two distinct epitopes underlying differential efficacy

doi: 10.1080/19420862.2026.2639673

Figure Lengend Snippet: Characterization of tozorakimab and related mutants. (A) Binding affinity of tozorakimab and mutants to IL33 was assessed by indirect ELISA. Wells were coated with IL33 (2 μg/mL). An anti-human IgG-HRP antibody (promega, cat. no. W4031 ) was used for detection. (B) Inhibition of IL-33-induced signaling by the tozorakimab and mutants. HEK-Blue IL-33 reporter cells were stimulated with IL-33 at 0.5 nM in the presence of a series of each antibody. After 22 hours, SEAP activity in the supernatant was quantified by measuring absorbance at 620 nm. Data points represent mean ± SD ( n = 3). Dose – response curves were fitted and EC 50 or IC 50 values were calculated using GraphPad prism ( n = 3); results are summarized in the right panel table.

Article Snippet: HEK-BlueTM IL-33 cells (InvivoGen, catalog no. hkb-hil33) were utilized for bioactivity assessment of IL-33 through monitoring NF-κB and AP-1 pathway activation.

Techniques: Binding Assay, Indirect ELISA, Inhibition, Activity Assay

Journal: mAbs

Article Title: Structures of clinical antibodies bound to IL-33 uncover two distinct epitopes underlying differential efficacy

doi: 10.1080/19420862.2026.2639673

Figure Lengend Snippet: SPR competition assays of three antibodies and ST2. IL-33 was immobilized on a CM5 sensor chip. Injections were performed in two steps: first, protein 1 was injected for 150 s, followed by a mixture of protein 1 and protein 2 for 150 s. From left to right: competition profiles for itepekimab + ST2, etokimab + ST2, and tozorakimab + ST2, ST2 = 200 nM，Abs = 100 nM.

Article Snippet: HEK-BlueTM IL-33 cells (InvivoGen, catalog no. hkb-hil33) were utilized for bioactivity assessment of IL-33 through monitoring NF-κB and AP-1 pathway activation.

Techniques: Injection

Journal: bioRxiv

Article Title: TAK1 integrates the NLRP1 inflammasome into the innate immune response to double-stranded RNA

doi: 10.64898/2026.01.23.701331

Figure Lengend Snippet: ( A-B ) Reconstitution of the NLRP1 inflammasome by co-transfecting 293T cells with plasmids encoding NLRP1, ASC, CASP1, and pro-IL-1β. Cells were treated with VbP (10μM), mock transfected, or co-transfected with HMW poly(I:C) (1μg/mL) or plasmids encoding ZAKα or CVB3 3C protease. Levels of bioactive IL-1β, reported as optical density (OD), were quantified using a HEK-Blue IL-1β reporter assay (A, see Methods). Immunoblotting of indicated proteins from lysates harvested 40-42 hours post-transfection (B). ( C-E ) NLRP1 inflammasome reconstituted 293T cells were co-transfected with empty vector, pRIG-I, pMDA5, or pTLR3 and treated with VbP (10μM), mock transfected, or transfected with HMW poly(I:C) (1μg/mL). Quantification of supernatant IL-1β levels (D) and immunoblotting (E) were carried out as previously described. Bioactive IFN-β levels were quantified using a HEK-Blue IFN reporter assay (C). ( F ) Schematic depicting drug (B/B) inducible murine RIG-I CARDs fused to FKBP12 F36V dimerizing domains (3xFV-N-RIG-I CARD ). ( G ) NLRP1 inflammasome reconstituted 293T cells were co-transfected with either an empty vector or the 3xFV-N-RIG-I CARD and treated with VbP (10μM) or B/B (10nM). IL-1β was quantified as in (A). Results are representative of two (A-B, E,), three (G), or four (C-D) independent experiments. Data are presented as mean ± SD. p-values were determined by two-way ANOVA with Tukey’s test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Supernatant IL-1β and type I IFN from 293T cells was quantified using the HEK-Blue IL-1β and IFN-α/β reporter cells (Invivogen), respectively.

Techniques: Transfection, Reporter Assay, Western Blot, Plasmid Preparation

Journal: bioRxiv

Article Title: TAK1 integrates the NLRP1 inflammasome into the innate immune response to double-stranded RNA

doi: 10.64898/2026.01.23.701331

Figure Lengend Snippet: ( A-B ) WT and indicated single or double knockout (KO) N/TERT-2G cells were primed with 10ng/mL TNFα for 24 hours and then treated with VbP (10μM), mock transfected, or transfected with HMW poly(I:C) (1μg/mL). Supernatants were harvested 24 hours following the addition of activating stimuli and IL-1β (A) and IFN-β (B) were measured using an antibody-based immunoassay (see Methods). ( C ) Immunoblotting for indicated proteins from lysates harvested 6 hours following mock or poly(I:C) transfection of unprimed WT and indicated KO N/TERT-2G cells. ( D-F ) NLRP1 reconstituted 293T cells were either treated with VbP (10μM) or co-transfected with an empty vector, pMAVS, or pTRIF. IL-1β (D) and IFN-β (E) levels were quantified as described in . Immunoblotting for indicated proteins was carried out as described in (C) from lysates harvested 40-42 hours post-transfection (F). Results are representative of two (A-C) or three (D-F) independent experiments. Data are presented as mean ± SD. p-values were determined by one-way ANOVA with Dunnett’s test (A-B) or by two-way ANOVA with Tukey’s test (D-E). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Supernatant IL-1β and type I IFN from 293T cells was quantified using the HEK-Blue IL-1β and IFN-α/β reporter cells (Invivogen), respectively.

Techniques: Double Knockout, Transfection, Western Blot, Plasmid Preparation

Journal: bioRxiv

Article Title: TAK1 integrates the NLRP1 inflammasome into the innate immune response to double-stranded RNA

doi: 10.64898/2026.01.23.701331

Figure Lengend Snippet: ( A-C ) N/TERT-2G cells were primed with 10ng/mL TNFα for 24 hours. Cells were incubated with either Ruxolitnib (Rux) (1μM) or IFN blocking antibody (anti-IFN) (1:50) for 1 hour prior to treatment with either VbP (10μM), recombinant IFN-β (rIFN-β), or HMW poly(I:C) (1μg/mL) and supernatants and cell pellets were harvested 10 hours post treatment. Expression of MX1, IFIT1 , and ISG15 in poly(I:C)-treated N/TERT-2G cells was determined by quantitative RT-PCR (A). IFN-β (B) and IL-1β (C) were quantified by ELISA. ( D-E ) WT and indicated KO N/TERT-2G cells were primed with 10ng/mL TNFα for 24 hours and treated with VbP (10μM) or HMW poly(I:C) (1μg/mL) for 24 hours. Supernatant IL-1β (D) and IFN-β (E) were quantified using an antibody-based immunoassay. ( F ) Immunoblotting from lysates generated from unprimed WT and indicated KO N/TERT-2G cells treated with VbP (10μM) or HMW poly(I:C) (1μg/mL) for 6 hours. Results are representative of two (A-F) independent experiments. Data are presented as mean ± SD. p-values were determined by one-way ANOVA with Dunnett’s test (A, D, and E) or two-way ANOVA with Sidak’s test (B-C). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Supernatant IL-1β and type I IFN from 293T cells was quantified using the HEK-Blue IL-1β and IFN-α/β reporter cells (Invivogen), respectively.

Techniques: Incubation, Blocking Assay, Recombinant, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Generated

Journal: bioRxiv

Article Title: TAK1 integrates the NLRP1 inflammasome into the innate immune response to double-stranded RNA

doi: 10.64898/2026.01.23.701331

Figure Lengend Snippet: ( A ) IL-1β levels were quantified by ELISA from supernatants of WT and indicated KO N/TERT-2G cells treated with VbP (10μM), ANS (1μM), or HMW poly(I:C) (1μg/mL) for 24 hours. ( B ) Immunoblotting for indicated proteins from lysates generated from N/TERT-2G cells treated as described in (A) 6 hours post-treatment. ( C ) Schematic depicting dsRNA activation of TAK1 and ribotoxic stress activation of ZAKa upstream of the NLRP1 inflammasome. ( D ) Primary keratinocytes from three independent donors were treated with Doramapimod (p38 inhibitor; 10μM) or Takinib (TAK1 inhibitor; 10μM) for 2 hours prior to treatment with VbP (10μM), ANS (1μM), or HMW poly(I:C) (1μg/mL) for 24 hours. Supernatant IL-1β levels were quantified by an antibody-based immunoassay. ( E-F ) NLRP1 reconstituted 293T cells were either treated with VbP (10μM) or co-transfected with pZAKα, pTAK1 WT, or pTAK1 K63W. Quantification of supernatant IL-1β using HEK-Blue IL-1β reporter cells (D). Immunoblotting for indicated proteins from lysates harvested 40-42 hours post-transfection (E). Results are representative of two (A-B) or three (D-F) independent experiments. Data are presented as mean ± SD. p-values were determined by two-way ANOVA with Sidak’s test (A, E) or by one-way ANOVA with Dunnett’s test (D) using GraphPad PRISM 10. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Supernatant IL-1β and type I IFN from 293T cells was quantified using the HEK-Blue IL-1β and IFN-α/β reporter cells (Invivogen), respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Generated, Activation Assay, Transfection

Journal: bioRxiv

Article Title: TAK1 integrates the NLRP1 inflammasome into the innate immune response to double-stranded RNA

doi: 10.64898/2026.01.23.701331

Figure Lengend Snippet: ( A ) Schematic depicting phosphorylation motifs in NLRP1’s N-terminal disordered region. ( B-C ) 293T cells were co-transfected with plasmids encoding MDA5 and WT or NLRP1 phophomotif mutants and treated with VbP (10μM) or HMW poly(I:C) (1μg/mL) (B and C) or co-transfected with plasmids encoding MAVS, TRIF, ZAKα, or TAK1 (C). ( D ) 293T cells were co-transfected with plasmids encoding MDA5 and WT NLRP1, WT CARD8, CARD8 NLRP1-DR , or CARD8 NLRP1-DR2×3A and treated with either treated with VbP (10μM) or HMW poly(I:C) (1μg/mL) or co-transfected plasmids encoding ZAKα or TAK1. Supernatant IL-1β levels were measured using an IL-1β bioassay (B-D). Results are representative of two (B and D) or three (C) independent experiments. Data are presented as mean ± SD. p-values were determined by two-way ANOVA with Tukey’s test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Supernatant IL-1β and type I IFN from 293T cells was quantified using the HEK-Blue IL-1β and IFN-α/β reporter cells (Invivogen), respectively.

Techniques: Phospho-proteomics, Transfection, Bioassay

Journal: bioRxiv

Article Title: TAK1 integrates the NLRP1 inflammasome into the innate immune response to double-stranded RNA

doi: 10.64898/2026.01.23.701331

Figure Lengend Snippet: ( A ) WT and indicated KO N/TERT-2G cells were treated with either VbP (10μM) or infected with either SINV AR86 or GW (MOI=20) for 48 hours. IL-1β was measured using an antibody-based immunoassay ( B ) Primary keratinocytes were treated with Doramapimod (10μM) or Takinib (10μM) for 2 hours prior to infection with SINV AR86 (MOI=20) for 48 hours. Supernatant IL-1β was measured as in (A). ( C-E ) WT and indicated KO N/TERT-2G cells were treated with siRNAs against ADAR or a non-targeting control (NTC) for 24 hours and primed with 10ng/mL TNFα. Supernatants were harvested 72 hours following siRNA treatment and IL-1β (E) and IFN-β (F) levels were measured using an antibody-based immunoassay. Immunoblotting for indicated proteins was carried out as described in (G) from lysates harvested 72 hours post transfection (G). Results are representative of two (A-E) independent experiments. Data are presented as mean ± SD. p-values were determined by one-way ANOVA with Dunnett’s test (A-B) or by two-way ANOVA with Sidak’s test (C-D) using GraphPad PRISM 10. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Supernatant IL-1β and type I IFN from 293T cells was quantified using the HEK-Blue IL-1β and IFN-α/β reporter cells (Invivogen), respectively.

Techniques: Infection, Control, Western Blot, Transfection