Journal: bioRxiv

Article Title: SLP2 coordinates MICOS assembly and cristae morphogenesis via MIC13 and YME1L

doi: 10.1101/2023.09.04.556207

Figure Lengend Snippet: A , Interactome of MIC13 with co-ip (co-immunoprecipitation) coupled mass spectrometry revealed SLP2 as a novel interactor of MIC13. B, The interaction between SLP2 and MIC13 was validated by co-ip using FLAG antibody in isolated mitochondria from MIC13 KO cells stably expressing MIC13-FLAG or empty vector (EV) pMSCVpuro as background control. I: input lanes represent loading of 10% of total lysates, E: eluate represent proteins eluted from anti-Flag M2 beads, U: unbound fraction. * non-specific IgG bands. C, Co-ip probed for SLP2-MICOS interaction with isolated mitochondria from SLP2 KO stably expressing pMSCVpuro EV (background control) or SLP2-MYC. Co-ip was performed using MYC-Trap agarose beads. I: Input fraction (10% of total lysate), E: Eluate fraction. YME1L was used as a positive interactor of SLP2 whereas Mt-CO2 and HSP60 served as non-interactors. All the MICOS subunits were present in the elution fraction from SLP2-MYC co-ip. D, Proximity ligation assay in HeLa cells with antibodies against MICOS subunits and SLP2. PLA signals are shown as red spots indicating respective protein interactions. SLP2 alone and Mt-CO2 & SLP2 antibodies were probed as negative controls.

Article Snippet: Human SLP2-HA ORF (Sino Biologicals, HG16147-CY) was cloned into pMSCVpuro vector using Gibson assembly cloning kit (NEB, E2611L), following manufacturer’s protocol.

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Mass Spectrometry, Isolation, Stable Transfection, Expressing, Plasmid Preparation, Proximity Ligation Assay

Journal: bioRxiv

Article Title: SLP2 coordinates MICOS assembly and cristae morphogenesis via MIC13 and YME1L

doi: 10.1101/2023.09.04.556207

Figure Lengend Snippet: A , Co-ip-western blot analysis from SLP2 KO or MIC10-subcomplex KO (left) and B , SLP2 KO or MIC60-subcomplex KO (right) stably expressing pMSCVpuro EV or SLP2-MYC showed that SLP2 can stably interact with any remaining MICOS subunits even upon the loss of individual MICOS subunits. Due to low abundance of MICOS proteins in MIC60 KO cells, overexposed blots were represented showing independent interaction of MIC13, MIC26, MIC27 and MIC19 with SLP2 in absence of MIC60. I: Input fraction (10% of total lysate), E: Eluate fraction. C, BN-PAGE with isolated mitochondria from WT cells revealed a co-migration pattern of SLP2 with higher molecular weight MICOS complex. D, Scaffolding model depicting interaction of SLP2 with MICOS subunits shows that SLP2 provides a scaffold for interaction of MICOS subunits.

Article Snippet: Human SLP2-HA ORF (Sino Biologicals, HG16147-CY) was cloned into pMSCVpuro vector using Gibson assembly cloning kit (NEB, E2611L), following manufacturer’s protocol.

Techniques: Co-Immunoprecipitation Assay, Western Blot, Stable Transfection, Expressing, Isolation, Migration, Molecular Weight, Scaffolding

Journal: bioRxiv

Article Title: SLP2 coordinates MICOS assembly and cristae morphogenesis via MIC13 and YME1L

doi: 10.1101/2023.09.04.556207

Figure Lengend Snippet: A , Steady state levels of MICOS proteins with western blot analysis from WT, SLP2 KO and SLP2 KO cells stably expressing pMSCVpuro EV or SLP2-MYC. B, BN-PAGE of isolated mitochondria from WT and SLP2 KO cells stained for MICOS subunits. C, Western blot analysis of steady state levels of MICOS proteins from WT and SLP2 KO cells stably expressing pGIPZ-control shRNA or YME1L shRNA (knockdown represented as KD). D, A model depicting the role of SLP2 in stabilizing MIC26 by regulating YME1L-mediated proteolysis. E, TEM images from WT, SLP2 KO and MIC26 KO cells. SLP2 KO shows accumulation of swollen cristae, while MIC26 KO shows interconnected cristae arranged in a honeycomb manner. F, Cristae number and CJs per mitochondrial section quantified from TEM images. Statistical analysis was performed with one-way ANOVA. *P-value ≤ 0.05, **P-value ≤ 0.01, ***P-value ≤ 0.001.

Article Snippet: Human SLP2-HA ORF (Sino Biologicals, HG16147-CY) was cloned into pMSCVpuro vector using Gibson assembly cloning kit (NEB, E2611L), following manufacturer’s protocol.

Techniques: Western Blot, Stable Transfection, Expressing, Isolation, Staining, shRNA

Journal: bioRxiv

Article Title: SLP2 coordinates MICOS assembly and cristae morphogenesis via MIC13 and YME1L

doi: 10.1101/2023.09.04.556207

Figure Lengend Snippet: A , Assessment of steady state levels of MICOS proteins with western blot from WT, MIC13 KO, SLP2 KO and MIC13-SLP2 DKO cells. B, BN-PAGE of isolated mitochondria from WT, MIC13 KO, SLP2 KO and MIC13-SLP2 DKO cells to assess MICOS assembly. MIC13-SLP2 DKO showed reduced MIC60 assembly in MICOS complex compared to any single KO. C, A model depicting that MIC60-subcomplex assembly is dependent on SLP2-MIC13 axis. D , TEM images displaying mitochondrial morphology from WT, MIC13 KO, SLP2 KO and MIC13-SLP2 DKO cells. E, Quantification of number of cristae and CJs per mitochondrial section obtained from TEM. *P-value ≤ 0.05, **P-value ≤ 0.01, ***P-value ≤ 0.001.

Article Snippet: Human SLP2-HA ORF (Sino Biologicals, HG16147-CY) was cloned into pMSCVpuro vector using Gibson assembly cloning kit (NEB, E2611L), following manufacturer’s protocol.

Techniques: Western Blot, Isolation

Journal: bioRxiv

Article Title: SLP2 coordinates MICOS assembly and cristae morphogenesis via MIC13 and YME1L

doi: 10.1101/2023.09.04.556207

Figure Lengend Snippet: A , WT cells stably expressing pLIX403 EV and MIC13 KO, MIC13-SLP2 DKO cells stably expressing pLIX403-MIC13-FLAG were treated with 1 μg/ml of doxycycline (Dox) for indicated time points and western blot analysis depicting steady state levels of MICOS proteins upon induction of MIC13-FLAG are shown. B, BN-PAGE with isolated mitochondria from WT cells stably expressing pLIX403 EV, and MIC13 KO and MIC13-SLP2 DKO cells stably expressing pLIX403-MIC13-FLAG treated with 1 μg/ml of doxycycline (Dox) for indicated time points showing stable incorporation of MIC13-FLAG in MICOS complex. C, Blue native PAGE with isolated mitochondria from WT cells stably expressing pLIX403 EV, and MIC13 KO and MIC13-SLP2 DKO cells stably expressing pLIX403-MIC13-FLAG treated with 1 μg/ml of Dox for indicated time points was probed for MIC10, MIC27 and MIC60 antibody. It shows that kinetics of MIC60 assembly was dependent on SLP2. D, A model depicting the assembly kinetics of MIC60 in MICOS complex depends on SLP2.

Article Snippet: Human SLP2-HA ORF (Sino Biologicals, HG16147-CY) was cloned into pMSCVpuro vector using Gibson assembly cloning kit (NEB, E2611L), following manufacturer’s protocol.

Techniques: Stable Transfection, Expressing, Western Blot, Isolation, Blue Native PAGE

Journal: bioRxiv

Article Title: SLP2 coordinates MICOS assembly and cristae morphogenesis via MIC13 and YME1L

doi: 10.1101/2023.09.04.556207

Figure Lengend Snippet: A , WT, MIC13 KO, SLP2 KO, MIC13-SLP2 DKO stably expressing pGIPZ-Control shRNA or pGIPZ-YME1L shRNA (knockdown represented as KD) subjected to western blot to assess steady state levels of MICOS proteins. The levels of MIC10, MIC26 and MIC27 were dependent on YME1L-mediated proteolysis. B, BN-PAGE with isolated mitochondria from WT, MIC13 KO, SLP2 KO, MIC13-SLP2 DKO stably expressing pGIPZ-Control shRNA or pGIPZ-YME1L shRNA. Red arrow indicates downshift of MIC60 and green arrow indicates upshift of MIC60 in BN-PAGE. C, Mitochondrial cristae morphology accessed using TEM from WT, MIC13 KO, SLP2 KO, MIC13-SLP2 DKO stably expressing pGIPZ-Control shRNA or pGIPZ-YME1L shRNA. Scale bar represents 0.5 µm. Red arrows depict CJs in the mitochondrial section showing a partial beneficial effect on cristae morphology upon YME1L depletion. D, Quantification of crista and CJs per mitochondrial section. Outliers were removed with Grubbs’ method and statistical significance was analysed by one-way ANOVA. *P-value ≤ 0.05, **P-value ≤ 0.01, ***P-value ≤ 0.001.

Article Snippet: Human SLP2-HA ORF (Sino Biologicals, HG16147-CY) was cloned into pMSCVpuro vector using Gibson assembly cloning kit (NEB, E2611L), following manufacturer’s protocol.

Techniques: Stable Transfection, Expressing, shRNA, Western Blot, Isolation

Journal: bioRxiv

Article Title: SLP2 coordinates MICOS assembly and cristae morphogenesis via MIC13 and YME1L

doi: 10.1101/2023.09.04.556207

Figure Lengend Snippet: A, STED nanoscopy images from WT, MIC13 KO, SLP2 KO and MIC13-SLP2 DKO cells displaying MIC60 punctae. White arrows indicate individual MIC60 punctae in a rail-like arrangement in WT cells. Arrow heads depict perturbed MIC60 punctae in MIC13 KO and SLP2 KO. Yellow arrow with curve depicts dispersed MIC60 punctae in MIC13-SLP2 DKO cells. B, A schematic model depicting the novel role of SLP2 to facilitate MICOS assembly and cristae morphogenesis via MIC13 and YME1L. The SLP2-YME1L and MIC13-YME1L axes control the quality and stability of the MIC10-subcomplex. The stable MIC10-subcomplex together with SLP2 was termed as amplifier complex, which promotes the incorporation of MIC60 into the MICOS-MIB complex. MIC60 is dispersed in the IBM upon the loss of SLP2 and MIC13. The amplifier complex provides the docking site for assembly of MIC60 and thus facilitates the MIC60 puncta formation. The model shows the co-dependence of MIC60- and MIC10-subcomplex for MICOS assembly and CJ formation.

Article Snippet: Human SLP2-HA ORF (Sino Biologicals, HG16147-CY) was cloned into pMSCVpuro vector using Gibson assembly cloning kit (NEB, E2611L), following manufacturer’s protocol.

Techniques:

Journal: bioRxiv

Article Title: SLP2 coordinates MICOS assembly and cristae morphogenesis via MIC13 and YME1L

doi: 10.1101/2023.09.04.556207

Figure Lengend Snippet: A , Interactome of MIC13 with co-ip (co-immunoprecipitation) coupled mass spectrometry revealed SLP2 as a novel interactor of MIC13. B, The interaction between SLP2 and MIC13 was validated by co-ip using FLAG antibody in isolated mitochondria from MIC13 KO cells stably expressing MIC13-FLAG or empty vector (EV) pMSCVpuro as background control. I: input lanes represent loading of 10% of total lysates, E: eluate represent proteins eluted from anti-Flag M2 beads, U: unbound fraction. * non-specific IgG bands. C, Co-ip probed for SLP2-MICOS interaction with isolated mitochondria from SLP2 KO stably expressing pMSCVpuro EV (background control) or SLP2-MYC. Co-ip was performed using MYC-Trap agarose beads. I: Input fraction (10% of total lysate), E: Eluate fraction. YME1L was used as a positive interactor of SLP2 whereas Mt-CO2 and HSP60 served as non-interactors. All the MICOS subunits were present in the elution fraction from SLP2-MYC co-ip. D, Proximity ligation assay in HeLa cells with antibodies against MICOS subunits and SLP2. PLA signals are shown as red spots indicating respective protein interactions. SLP2 alone and Mt-CO2 & SLP2 antibodies were probed as negative controls.

Article Snippet: Following primary antibodies were used: MIC10 (Abcam, 84969), MIC13 (custom made by Pineda (Berlin) against human MIC13 peptide CKAREYSKEGWEYVKARTK), MIC19 (Proteintech, 25625-1-AP), MIC25 (Proteintech, 20639-1-AP), MIC26 (Thermofisher Scientific, MA5-15493), MIC27 (Sigma-Aldrich, HPA000612-100UL), MIC60 (Abcam, ab110329), SLP2 (Abcam, ab102051), SLP2 (OriGene, TA808240), Mt-CO2 (Abcam, ab110258).

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Mass Spectrometry, Isolation, Stable Transfection, Expressing, Plasmid Preparation, Control, Proximity Ligation Assay

Journal: bioRxiv

Article Title: SLP2 coordinates MICOS assembly and cristae morphogenesis via MIC13 and YME1L

doi: 10.1101/2023.09.04.556207

Figure Lengend Snippet: A , Co-ip-western blot analysis from SLP2 KO or MIC10-subcomplex KO (left) and B , SLP2 KO or MIC60-subcomplex KO (right) stably expressing pMSCVpuro EV or SLP2-MYC showed that SLP2 can stably interact with any remaining MICOS subunits even upon the loss of individual MICOS subunits. Due to low abundance of MICOS proteins in MIC60 KO cells, overexposed blots were represented showing independent interaction of MIC13, MIC26, MIC27 and MIC19 with SLP2 in absence of MIC60. I: Input fraction (10% of total lysate), E: Eluate fraction. C, BN-PAGE with isolated mitochondria from WT cells revealed a co-migration pattern of SLP2 with higher molecular weight MICOS complex. D, Scaffolding model depicting interaction of SLP2 with MICOS subunits shows that SLP2 provides a scaffold for interaction of MICOS subunits.

Article Snippet: Following primary antibodies were used: MIC10 (Abcam, 84969), MIC13 (custom made by Pineda (Berlin) against human MIC13 peptide CKAREYSKEGWEYVKARTK), MIC19 (Proteintech, 25625-1-AP), MIC25 (Proteintech, 20639-1-AP), MIC26 (Thermofisher Scientific, MA5-15493), MIC27 (Sigma-Aldrich, HPA000612-100UL), MIC60 (Abcam, ab110329), SLP2 (Abcam, ab102051), SLP2 (OriGene, TA808240), Mt-CO2 (Abcam, ab110258).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Stable Transfection, Expressing, Isolation, Migration, Molecular Weight, Scaffolding

Journal: bioRxiv

Article Title: SLP2 coordinates MICOS assembly and cristae morphogenesis via MIC13 and YME1L

doi: 10.1101/2023.09.04.556207

Figure Lengend Snippet: A , Steady state levels of MICOS proteins with western blot analysis from WT, SLP2 KO and SLP2 KO cells stably expressing pMSCVpuro EV or SLP2-MYC. B, BN-PAGE of isolated mitochondria from WT and SLP2 KO cells stained for MICOS subunits. C, Western blot analysis of steady state levels of MICOS proteins from WT and SLP2 KO cells stably expressing pGIPZ-control shRNA or YME1L shRNA (knockdown represented as KD). D, A model depicting the role of SLP2 in stabilizing MIC26 by regulating YME1L-mediated proteolysis. E, TEM images from WT, SLP2 KO and MIC26 KO cells. SLP2 KO shows accumulation of swollen cristae, while MIC26 KO shows interconnected cristae arranged in a honeycomb manner. F, Cristae number and CJs per mitochondrial section quantified from TEM images. Statistical analysis was performed with one-way ANOVA. *P-value ≤ 0.05, **P-value ≤ 0.01, ***P-value ≤ 0.001.

Article Snippet: Following primary antibodies were used: MIC10 (Abcam, 84969), MIC13 (custom made by Pineda (Berlin) against human MIC13 peptide CKAREYSKEGWEYVKARTK), MIC19 (Proteintech, 25625-1-AP), MIC25 (Proteintech, 20639-1-AP), MIC26 (Thermofisher Scientific, MA5-15493), MIC27 (Sigma-Aldrich, HPA000612-100UL), MIC60 (Abcam, ab110329), SLP2 (Abcam, ab102051), SLP2 (OriGene, TA808240), Mt-CO2 (Abcam, ab110258).

Techniques: Western Blot, Stable Transfection, Expressing, Isolation, Staining, Control, shRNA, Knockdown

Journal: bioRxiv

Article Title: SLP2 coordinates MICOS assembly and cristae morphogenesis via MIC13 and YME1L

doi: 10.1101/2023.09.04.556207

Figure Lengend Snippet: A , Assessment of steady state levels of MICOS proteins with western blot from WT, MIC13 KO, SLP2 KO and MIC13-SLP2 DKO cells. B, BN-PAGE of isolated mitochondria from WT, MIC13 KO, SLP2 KO and MIC13-SLP2 DKO cells to assess MICOS assembly. MIC13-SLP2 DKO showed reduced MIC60 assembly in MICOS complex compared to any single KO. C, A model depicting that MIC60-subcomplex assembly is dependent on SLP2-MIC13 axis. D , TEM images displaying mitochondrial morphology from WT, MIC13 KO, SLP2 KO and MIC13-SLP2 DKO cells. E, Quantification of number of cristae and CJs per mitochondrial section obtained from TEM. *P-value ≤ 0.05, **P-value ≤ 0.01, ***P-value ≤ 0.001.

Article Snippet: Following primary antibodies were used: MIC10 (Abcam, 84969), MIC13 (custom made by Pineda (Berlin) against human MIC13 peptide CKAREYSKEGWEYVKARTK), MIC19 (Proteintech, 25625-1-AP), MIC25 (Proteintech, 20639-1-AP), MIC26 (Thermofisher Scientific, MA5-15493), MIC27 (Sigma-Aldrich, HPA000612-100UL), MIC60 (Abcam, ab110329), SLP2 (Abcam, ab102051), SLP2 (OriGene, TA808240), Mt-CO2 (Abcam, ab110258).

Techniques: Western Blot, Isolation

Journal: bioRxiv

Article Title: SLP2 coordinates MICOS assembly and cristae morphogenesis via MIC13 and YME1L

doi: 10.1101/2023.09.04.556207

Figure Lengend Snippet: A , WT cells stably expressing pLIX403 EV and MIC13 KO, MIC13-SLP2 DKO cells stably expressing pLIX403-MIC13-FLAG were treated with 1 μg/ml of doxycycline (Dox) for indicated time points and western blot analysis depicting steady state levels of MICOS proteins upon induction of MIC13-FLAG are shown. B, BN-PAGE with isolated mitochondria from WT cells stably expressing pLIX403 EV, and MIC13 KO and MIC13-SLP2 DKO cells stably expressing pLIX403-MIC13-FLAG treated with 1 μg/ml of doxycycline (Dox) for indicated time points showing stable incorporation of MIC13-FLAG in MICOS complex. C, Blue native PAGE with isolated mitochondria from WT cells stably expressing pLIX403 EV, and MIC13 KO and MIC13-SLP2 DKO cells stably expressing pLIX403-MIC13-FLAG treated with 1 μg/ml of Dox for indicated time points was probed for MIC10, MIC27 and MIC60 antibody. It shows that kinetics of MIC60 assembly was dependent on SLP2. D, A model depicting the assembly kinetics of MIC60 in MICOS complex depends on SLP2.

Article Snippet: Following primary antibodies were used: MIC10 (Abcam, 84969), MIC13 (custom made by Pineda (Berlin) against human MIC13 peptide CKAREYSKEGWEYVKARTK), MIC19 (Proteintech, 25625-1-AP), MIC25 (Proteintech, 20639-1-AP), MIC26 (Thermofisher Scientific, MA5-15493), MIC27 (Sigma-Aldrich, HPA000612-100UL), MIC60 (Abcam, ab110329), SLP2 (Abcam, ab102051), SLP2 (OriGene, TA808240), Mt-CO2 (Abcam, ab110258).

Techniques: Stable Transfection, Expressing, Western Blot, Isolation, Blue Native PAGE

Journal: bioRxiv

Article Title: SLP2 coordinates MICOS assembly and cristae morphogenesis via MIC13 and YME1L

doi: 10.1101/2023.09.04.556207

Figure Lengend Snippet: A , WT, MIC13 KO, SLP2 KO, MIC13-SLP2 DKO stably expressing pGIPZ-Control shRNA or pGIPZ-YME1L shRNA (knockdown represented as KD) subjected to western blot to assess steady state levels of MICOS proteins. The levels of MIC10, MIC26 and MIC27 were dependent on YME1L-mediated proteolysis. B, BN-PAGE with isolated mitochondria from WT, MIC13 KO, SLP2 KO, MIC13-SLP2 DKO stably expressing pGIPZ-Control shRNA or pGIPZ-YME1L shRNA. Red arrow indicates downshift of MIC60 and green arrow indicates upshift of MIC60 in BN-PAGE. C, Mitochondrial cristae morphology accessed using TEM from WT, MIC13 KO, SLP2 KO, MIC13-SLP2 DKO stably expressing pGIPZ-Control shRNA or pGIPZ-YME1L shRNA. Scale bar represents 0.5 µm. Red arrows depict CJs in the mitochondrial section showing a partial beneficial effect on cristae morphology upon YME1L depletion. D, Quantification of crista and CJs per mitochondrial section. Outliers were removed with Grubbs’ method and statistical significance was analysed by one-way ANOVA. *P-value ≤ 0.05, **P-value ≤ 0.01, ***P-value ≤ 0.001.

Article Snippet: Following primary antibodies were used: MIC10 (Abcam, 84969), MIC13 (custom made by Pineda (Berlin) against human MIC13 peptide CKAREYSKEGWEYVKARTK), MIC19 (Proteintech, 25625-1-AP), MIC25 (Proteintech, 20639-1-AP), MIC26 (Thermofisher Scientific, MA5-15493), MIC27 (Sigma-Aldrich, HPA000612-100UL), MIC60 (Abcam, ab110329), SLP2 (Abcam, ab102051), SLP2 (OriGene, TA808240), Mt-CO2 (Abcam, ab110258).

Techniques: Stable Transfection, Expressing, Control, shRNA, Knockdown, Western Blot, Isolation

Journal: bioRxiv

Article Title: SLP2 coordinates MICOS assembly and cristae morphogenesis via MIC13 and YME1L

doi: 10.1101/2023.09.04.556207

Figure Lengend Snippet: A, STED nanoscopy images from WT, MIC13 KO, SLP2 KO and MIC13-SLP2 DKO cells displaying MIC60 punctae. White arrows indicate individual MIC60 punctae in a rail-like arrangement in WT cells. Arrow heads depict perturbed MIC60 punctae in MIC13 KO and SLP2 KO. Yellow arrow with curve depicts dispersed MIC60 punctae in MIC13-SLP2 DKO cells. B, A schematic model depicting the novel role of SLP2 to facilitate MICOS assembly and cristae morphogenesis via MIC13 and YME1L. The SLP2-YME1L and MIC13-YME1L axes control the quality and stability of the MIC10-subcomplex. The stable MIC10-subcomplex together with SLP2 was termed as amplifier complex, which promotes the incorporation of MIC60 into the MICOS-MIB complex. MIC60 is dispersed in the IBM upon the loss of SLP2 and MIC13. The amplifier complex provides the docking site for assembly of MIC60 and thus facilitates the MIC60 puncta formation. The model shows the co-dependence of MIC60- and MIC10-subcomplex for MICOS assembly and CJ formation.

Article Snippet: Following primary antibodies were used: MIC10 (Abcam, 84969), MIC13 (custom made by Pineda (Berlin) against human MIC13 peptide CKAREYSKEGWEYVKARTK), MIC19 (Proteintech, 25625-1-AP), MIC25 (Proteintech, 20639-1-AP), MIC26 (Thermofisher Scientific, MA5-15493), MIC27 (Sigma-Aldrich, HPA000612-100UL), MIC60 (Abcam, ab110329), SLP2 (Abcam, ab102051), SLP2 (OriGene, TA808240), Mt-CO2 (Abcam, ab110258).

Techniques: Control

Journal: The Lancet. Rheumatology

Article Title: Autoantibodies against interleukin-1 receptor antagonist in multisystem inflammatory syndrome in children: a multicentre, retrospective, cohort study

doi: 10.1016/S2665-9913(22)00064-9

Figure Lengend Snippet: Neutralising and functional effect of anti-IL-1Ra antibodies in MIS-C (A) Free IL-1Ra plasma concentrations as measured by ELISA in patients with MIS-C (n=21), Kawasaki disease (n=6) and systemic juvenile idiopathic arthritis (n=10). Horizontal lines represent the mean and SD. Data were analysed by Brown–Forsythe and Welch ANOVA with Dunnett's T3 multiple comparisons. (B) IL-1β-signalling reporter assay on selected MIS-C plasma compared with an adult critical COVID-19 plasma sample (both 1:20 dilution) as well as commercially available anti-IL-1Ra antibody or control (anti-SLP2) antibody. The absorbance of secreted embryonic alkaline phosphatase, as a marker for IL-1β pathway activation in HEK IL-1β reporter cells, was detected at 650 nm. Error bars show mean (SD). MIS-C=multisystem inflammatory syndrome in children. IL-1Ra=interleukin-1 receptor antagonist. KD=Kawasaki disease. sJIA=systemic juvenile idiopathic arthritis. TNF=tumour necrosis factor. IL-1β=interleukin-1β.

Article Snippet: Recombinant IL-1Ra at 40 ng/mL (Biozol, Eching, Germany) alone or with either rabbit antihuman IL-1Ra antibody at 5 μg/mL (antibodies-online, Aachen, Germany), recombinant anti-SLP2 antibody at 5 μg/mL (Abcam, Cambridge, UK), diluted plasma (1:20) from a patient with acute MIS-C and high-titred (1:800) anti-IL-1Ra antibodies, or diluted plasma (1:20) from the same patient 7 months after onset of MIS-C but without detectable anti-IL-1Ra antibodies, were preincubated for 2 h at room temperature.

Techniques: Functional Assay, Enzyme-linked Immunosorbent Assay, Reporter Assay, Marker, Activation Assay