EMBO/soda-nel huggingface dataset:
In vivo molecular imaging of chemokine receptor CXCR4 expression in patients with advanced multiple myeloma. (2015)
Figure 1

CXCR4 expression in MM patient bone marrow

Flow cytometric evaluation of CXCR4 surface expression using an anti-CXCR4-PE antibody. Left: positive patient; right: negative patient; representative data are shown. The gating strategy is depicted in Supplementary Fig S1.

Representative histograms revealing the magnitude of CXCR4 expression in myeloma cells as compared to the indicated bone marrow cell subtype.

Median fluorescence intensity of surface CXCR4 expression relative to isotype control (n = 7–10 patients). Horizontal bars indicate mean of all individual patient values; asterisks indicate statistical significance (plasma cells vs B cells: P = 0.0082; plasma cells vs T cells: P = 0.0091; plasma cells vs CD34+ cells: P = 0.011; plasma cells vs monocytes: P = 0.0423; Student's t-test to compare mean plasma cell relative expression with mean relative expression of each further indicated cell subtype). MM patients judged positive for CXCR4 expression were selected for this analysis.

Representative MM bone marrow staining of a patient positive for MM CXCR4 expression: hematoxylin and eosin (H&E) staining. Immunohistochemistry for CD138, CXCR4, light chain kappa, and light chain lambda.
In vivo molecular imaging of chemokine receptor CXCR4 expression in patients with advanced multiple myeloma. (2015)
Figure 2

[<sup>68</sup>Ga]Pentixafor PET imaging of MM xenografts

Real-time PCR analysis of cxcr4 transcript expression levels in HeLa (negative control) and in MM cell lines MM.1S and OPM-2. Shown is the mean relative expression ± SEM (n = 3 independent experiments). Values are normalized to the expression of ubiquitin (Ub). The asterisk indicates statistically significant differences (HeLa vs MM.1S: P < 0.001; HeLa vs OPM-2: P < 0.0001; Student's t-test).

CXCR4 protein expression assessed by immunoblotting (one representative blot out of 3 is shown).

Binding of [68Ga]Pentixafor to MM.1S and OPM-2 cells after the indicated incubation periods (n = 3 per cell line and time point). Shown is the mean ± SEM. The difference between the OPM-2 groups is statistically significant; *P < 0.0017 (one-way ANOVA).

Mean tumor-to-background ratio (TBR) for [18F]FDG (left) and for [68Ga]Pentixafor (right) in MM.1S and OPM-2 xenograft-bearing NOD SCID mice. Shown is the mean ± SEM, n = 8 tumors (4 mice); *P = 0.0111 for [18F]FDG and *P = 0.0113 for [68Ga]Pentixafor (Student's t-test). One-way ANOVA revealed significant differences between the groups; P < 0.0001 (not graphically shown).

Representative [68Ga]Pentixafor PET images of three mice bearing MM.1S (right shoulder) and OPM-2 (left shoulder) tumors.

Flow cytometric quantification of CXCR4 cell surface expression on resected MM.1S and OPM-2 tumors. Data are the mean ± SEM, n = 4. *P = 0.0286; Mann–Whitney U-test.

Correlation of [68Ga]Pentixafor PET mean TBR and CXCR4 cell surface expression assessed by flow cytometry. n = 8 tumors were analyzed.

Mice (n = 4) bearing OPM-2 and MM.1S xenografts were coinjected with AMD3100 (right image, one representative mouse) or not pretreated (left image) before undergoing [68Ga]Pentixafor PET. The white arrows point to the bladder. Quantification is shown in Supplementary Fig S3A.

Source data are available online for this figure.
Unfolded protein response IRE1/XBP1 signaling is required for healthy mammalian brain aging (2022)
Figure 1

Figure 1. Impaired UPR activation in the mammalian aged brain.

(A) Functional enrichment analysis of altered genes in an elderly cohort of patients diagnosed with dementia versus non-demented. GO: gene ontology; Key TF: key transcription factor in TRRUST database; FDR: false discovery rate.

(B) Violin plots displaying RNA-seq readings by FPKM (normalized Fragments Per Kilobase Million) of chaperones HSPA1A and HSPA1B of human hippocampus samples of demented patients and controls (n = 49,42; unpaired Kolmogorov-Smirnov test; continuous lines: median; dashed lines: quartiles).

(C) Pharmacological models of ER stress induction. Animals of indicated ages were injected with ER stress inducer tunicamycin through intraperitoneal (i.p.) injection. After 24 h, the brain was dissected to measure UPR signaling using RT-PCR. Alternatively, animals received tunicamycin intracerebrally (i.c.) directly into the hippocampus using stereotaxic injection.

(D) Mean and SEM of mRNA levels of Xbp1s, Bip/Hspa5 and Chop/Ddit3 were determined in dissected hippocampus of i.p. treated mice by quantitative RT-PCR. (n = 3-4 animals/group. One-way ANOVA followed by Tukey's post-test compared tunicamycin treated groups).

(E) Animals received bilateral intra-hippocampal injections of 2 μl tunicamcyin (5 mg/mL) or vehicle for 24 h. Graphs indicate mean and s.e.m. of Xbp1s mRNA levels in the hippocampus (n = 3-4 animals/group. One-way ANOVA followed by Tukey's post-test compared tunicamycin treated groups.

(F-G) Mean and SEM of relative levels of S-nitrosylated IRE1 (f) and total IRE1 (g) in brains from young and aged mice. Histograms show relative levels of SNO-IRE1/input IRE1α measured by biotin-switch assay and total IRE1/GAPDH quantified by densitometry on standard immunoblots for young (3-month-old) versus aged (24 month-old) mouse brains (n = 3, unpaired Student's t test). Data information: All graphs indicate P values with the following legend: * = P < 0.05; ** = P <0.01.
Unfolded protein response IRE1/XBP1 signaling is required for healthy mammalian brain aging (2022)
Figure 2

Figure 2. Genetic ablation of the IRE1 pathway in the brain accelerates and exacerbates age-associated cognitive decline in mammals.

(A-D) Young (3-6 months), middle aged (12-14 months) and aged (18-20 months) conditional knockout for IRE1 (IRE1cKO) and their littermates flox/flox control animals (IRE1WT) were evaluated in new object recognition test (A), and contextual fear conditioning test (B); (please refer to Appendix Figures for complete workflow). (A) Mean and s.e.m. of the difference in exploration time between novel and familiar objects divided by the total amount of exploration time of both objects (%). n = 15,18;10,16;10,10. Two-way ANOVA followed by Holm-Sidak's multiple comparison test; P values: Age = 0.0001; Genotype = 0.02; Interaction = 0.40. (B) Mean and s.e.m. of percentage of time of freezing in the first minute before sound presentation in test day (day 2). N =7,11;9, 15;10, 8. Two-way ANOVA followed by Holm-Sidak's multiple comparison test; P values: Age =0.0341; Genotype=0.87; Interaction =0.0018. (C) Representative photomicrographs of β-galactosidase staining of hippocampal slices derived from young (3 months), middle-aged (12 months) and aged (22-24 months) of IRE1WT or IRE1cKO animals. Magnification: 20x; scale bar: 200 μm. Arrows indicate β-galactosidase staining. (D) Mean and SEM of percentage of β-galactosidase staining in CA3 region (n = 3-5 animals/group). P computed by unpaired Student's t test within each age-matched group

(E) Young (3 months) and middle aged (12 months) IRE1 flox/flox (IRE1WT) mice were evaluated in the new object recognition test before and after intra-hippocampal injections with AAV2-Cre. Controls were injected with AAV2-Mock. Graph indicates mean and SEM of the difference in exploration time between novel and familiar objects divided by the total amount of exploration time of both objects (%) before and after treatment. n = 12, 12, 8, Two-way ANOVA with repeated measurements followed by Holm-Sidak's multiple comparison test; P values: AAV-Cre treatment: 0.0001; Age = 0.08; Interaction = 0.0053.

(F) Representative confocal images of dendritic spines (arrows) in the CA1 region of middle-aged IRE1WT animals injected with AAV-Mock or AAV-Cre. 60x magnification, scale bar: 5μm.

(G) Mean and SEM of spine density per μm (n = 3, 3; 4, 4 animals). P computed by unpaired Student's t test within each age-matched group.

Data information: All graphs indicate P values with the following legend: * = P < 0.05; ** = P <0.01; *** = P < 0.005.
Unfolded protein response IRE1/XBP1 signaling is required for healthy mammalian brain aging (2022)
Figure 3

Figure 3. Enforced expression of XBP1s in the brain prevents age-associated cognitive decline in mammals.

(A-D) Young (3-6 months), middle aged (12-14 months) and aged (18-20 months) TgXBP1s and their littermate control animals Non Tg were evaluated in new object location (A), new object recognition (B), Barnes Maze (c-d). Please refer to Data S3 and S5 for complete workflow. (A) Mean and s.e.m. of the difference in exploration time between displaced and familiar objects divided by the total amount of exploration time of both objects (%). n = 10, 9; 17, 16; 12, 11; Two-way ANOVA followed by Holm-Sidak's multiple comparison test; P (Interaction between Age and Genotype) = 0.0048. (B) Mean and SEM of the difference in exploration time between novel and familiar objects divided by the total amount of exploration time of both objects (%). n = 16, 12; 14, 10; 16, 14; Two-way ANOVA followed by Holm-Sidak's multiple comparison test; P (Interaction between Age and Genotype) = 0.0186. (C) Mean and SEM of latencies to find the target hole in the Barnes maze test during 4 days of training comparing middle-aged (12 months) and aged (20-22 months) TgXBP1s with littermate controls. n = 10, 5; 7, 7; 8, 10; Two-way ANOVA with repeated measurements followed by Holm-Sidak's multiple comparison test; P (Genotype) = 0.0004. (D) Mean and SEM of latencies to find the target hole in test day. n =10, 5; 7, 7; 8, 10; Two-way ANOVA followed by Holm-Sidak's multiple comparison test; P values: Age = 0.0013; Genotype < 0.0001; Interaction = 0.0042.

(E) Representative images of β-galactosidase staining of hippocampal slices derived from aged (22-24 months) TgXBP1s and WT animals. Magnification: 20x; scale bar: 200 μm.

(F) Mean and SEM of percentage of area stained by β-galactosidase in the hippocampus of aged (20-24 months) TgXBP1s compared to littermate controls. n= 8, 5; unpaired Student's t test.

Data information: All graphs indicate P values with the following legend: * = P < 0.05; ** = P <0.01; *** = P < 0.005; **** = P < 0.001.
MCUR1 is an essential component of mitochondrial Ca2+ uptake that regulates cellular metabolism (2012)
figf1

(a,b) Changes in HEK293T cell cytoplasmic (a) and mitochondrial (b) [Ca2+] in response to ionomycin (2.5 μM) were simultaneously measured by fluo-4 and rhod-2 imaging, respectively. Each bar represents one target gene silenced with pooled siRNA. (c) qrtPCR of MCU, MCUR1 and MICU1 mRNA from mouse tissues (n = 3; mean ± s.e.m.). (d) qrtPCR of MCUR1 mRNA from HEK293T cell clones (n = 3; mean ± s.e.m.). KD, knockdown. Neg shRNA, negative shRNA. (e) qrtPCR of MCUR1 mRNA from HeLa cell clones and of rescued MCUR1 mRNA levels in shHe2 clone cells (n = 3; mean ± s.e.m.). The same lentiviral shRNAs were used to generate shHK4 and shHe1 and shHK5 and shHe2, respectively. (f) MCUR1 protein expression levels (top) and densitometric analysis (bottom left) (n = 3; ± s.e.m.). Bottom right, Flag-tagged MCUR1 protein expression in clone shHe2 cells reconstituted with shRNA-resistant MCUR1 cDNA plasmid. WB, western blot. (g,h) Representative images from movies of HEK293T negative shRNA or shHK5 cells showing cytosolic (green) and mitochondrial (red) [Ca2+] before, during and after ionomycin exposure. Scale bar, 20 μm. (i–p) Cytoplasmic (green) and mitochondrial matrix (red) [Ca2+] responses in HEK293T (i–l) and HeLa (m–p) cells challenged with ionomycin or histamine (100 μM), respectively. (n = 3). (i) Wild-type HEK293T cells. (j) Cells expressing negative shRNA. (k) Clone shHK5 (n = 4). (l) Quantification of peak rhod-2 fluorescence. **P0.01 (mean±s.e.m.). (m) HeLa cells expressing negative shRNA. (n) Clone shHe2. (o) Clone shHe2 re-expressing MCUR1 (n = 3). (p) Quantification of peak rhod-2 fluorescence. *P0.05, **P0.01 (mean±s.e.m.). (q) [Ca2+]c and [Ca2+]m signals evoked by ATP (100 μM) and thapsigargin (Tg, 2 μM) were monitored simultaneously using fura-2/AM and mitopericam (ipcam490), respectively in control (left) and MCUR1-knockdown (right) HeLa cells. [Ca2+]c is calibrated in nanomolar concentrations (black), whereas mitopericam fluorescence is inversely normalized to baseline (F0/F; red). (r) Summary mean [Ca2+]c and [Ca2+]m peaks during ATP stimulation (negative shRNA n = 29; MCUR1 knockdown n = 36 cells). *P0.05 (mean±s.e.m.). (s) Increase in bath [Ca2+](Rfura2) and [Ca2+]m (Ripcam) signals in response to CaCl2 (1 μM) and InsP3 (IP3, 7.5 μM) addition in permeabilized cells. Uncropped images of blots/gels are shown in Supplementary Fig. S6.
MCUR1 is an essential component of mitochondrial Ca2+ uptake that regulates cellular metabolism (2012)
figf2

Digitonin-permeabilized HeLa cells bathed in intracellular-like solution containing thapsigargin (Tg) were loaded with the ΔΨm indicator JC-1 and the Ca2+ indicator Fura2FF, to which pulses of 10 μM Ca2+ were added before the addition of the mitochondrial uncoupler CCCP (carbonyl cyanide m-chloro phenyl hydrazone). (a–e) Representative traces from three independent experiments depict simultaneous changes of bath [Ca2+] and ΔΨm in cells expressing negative shRNA (Neg shRNA; a), clone shHe1 (b), clone shHe2 (c), clone shHe2 re-expressing MCUR1 (d) and HeLa cells stably overexpressing MCUR1 (e). Under similar conditions, 1 μM Ru360 was added before 10 μM Ca2+ pulses until the addition of CCCP. (f–i) Representative traces from three independent experiments depict simultaneous changes of bath [Ca2+] and ΔΨm in negative shRNA cells (f), MCUR1-knockdown clone shHe1 (g) and shHe2 (h), and in control cells overexpressing MCUR1 (i). (j,k) Negative shRNA (j) and MCUR1-overexpressing (k) HEK293T cells were permeabilized with digitonin in intracellular-like medium containing thapsigargin and bath [Ca2+] indicator Fura2FF, and then pulsed with 10 μM Ca2+. After mitochondrial clearance of bath Ca2+, Ru360 caused an elevation of bath [Ca2+], indicating that steady-state bath [Ca2+] after the pulse was maintained by a balance of MCU-mediated Ca2+ uptake and CGP37157 (10 μM)-sensitive Na+–Ca2+ exchanger-mediated extrusion. CCCP was added as indicated. The solid line is the mean; shaded areas are ±s.e.m. (n = 3). (l) [Ca2+]m efflux rate derived from j and k during the initial 60 s following Ru360 addition (NS; not significant; n = 3). (m) HEK293T cells stably expressing negative shRNA or MCUR1 shRNA (clone shHe2) and clone shHe2 re-expressing MCUR1 cells were permeabilized with digitonin in intracellular-like medium containing the bath [Ca2+] indicator Fura2FF. CCCP was added as indicated. Traces show bath [Ca2+] (μM). Solid lines are the mean; shaded regions are ±s.e.m. (n =  3). (n) Quantification of total mitochondrial Ca2+ released after CCCP addition. *P0.05, **P0.01 (mean±s.e.m.).
MCUR1 is an essential component of mitochondrial Ca2+ uptake that regulates cellular metabolism (2012)
figf3

(a,b) Confocal images of HeLa cells transiently co-transfected for 48 h with GFP-tagged MCUR1 and DsRed-Mito plasmids (a) or mRFP-tagged MCUR1 and EYFP–Mito (b). Scale bar, 20 μm. (c) Immunoblot analysis of Flag-tagged MCUR1 in the HeLa cell crude mitochondrial fraction, outer mitochondrial membrane (OMM) and mitoplasts, using antibodies against VDAC1 (outer mitochondrial membrane protein), COII (integral membrane marker) and Flag. (d) Immunoblot analyses of mitochondria-containing pellet and cytosolic fractions from plasma-membrane-permeabilized HeLa cells. Permeabilized cells were treated with or without tBid (50 nM) for outer mitochondrial membrane permeabilization and the appearance of cytosolic cytochrome c was verified. Intact and outer mitochondrial membrane permeabilized samples were exposed to proteinase K for 10 min. These samples were probed using antibodies against HSP60, OXA1, Flag and MCUR1. (e) Reciprocal co-immunoprecipitation (IP) of MCU–GFP and MCUR1–Flag transiently expressed in COS7 cells. Representative of four independent experiments. WB, western blot. (f) Co-immunoprecipitation of MICU1–Flag with MCU–GFP but not with MCUR1–GFP transiently expressed in COS7 cells. Representative of four independent experiments. (g) Immunoprecipitation of LETM1–GFP with anti-LETM1 failed to pull down MCUR1–Flag in transiently transfected COS7 cells. (h) Immunoprecipitation with Flag antibody pulled down LETM1–Flag or MCU–Flag transiently expressed in COS7 cells (immunoprecipitation lanes 2 and 3, lower panel), but only co-immunoprecipitated MCUR1–V5 in the MCU–Flag-expressing cells (immunoprecipitation lanes 4 versus 5, upper panel), despite lower expression of MCU–Flag in MCUR1-co-transfected cells (compare MCU–Flag and LETM1–Flag expression in MCUR1-co-transfected cells in lysate lanes 4 and 5, bottom panel). Representative of three independent experiments. Uncropped images of blots/gels are shown in Supplementary Fig. S6.
MCUR1 is an essential component of mitochondrial Ca2+ uptake that regulates cellular metabolism (2012)
figf4

(a) qrtPCR of MCU mRNA from wild-type and stable MCUR1-overexpressing HeLa cells that were transiently transfected with scrambled siRNA or siRNA against MCU. ***P0.001 (mean±s.e.m.). (b) [Ca2+]m responses to histamine (100 μM) in HeLa cells stably overexpressing MCUR1 and in cells transiently transfected with scrambled siRNA or MCU siRNA, and in stable MCUR1-overexpressing HeLa cells transfected with MCU siRNA. After 48 h of siRNA transfection, cells were loaded with rhod-2 and [Ca2+]m responses were visualized by confocal microscopy. Solid lines are mean; shaded regions are ±s.e.m.; n = 3. (c) Quantification of peak rhod-2 fluorescence following histamine stimulation. *P0.05, ***P0.001 (mean±s.e.m.; NS, not significant; n = 3). (d) qrtPCR of MCU mRNA from wild-type and MCUR1-knockdown HeLa cells that were transiently transfected with MCU cDNA. **P0.01, ***P0.001 (mean±s.e.m.; n = 3). (e) [Ca2+]m responses to histamine (100 μM) in wild-type and MCUR1 (shHe2)-knockdown HeLa cells overexpressing MCU. Negative shRNA (Neg shRNA) and MCUR1-shHe2 cells were used as controls. Solid lines are mean; shaded regions are ±s.e.m.; n = 3. (f) Quantification of peak rhod-2 fluorescence following histamine stimulation. ***P0.001 (mean±s.e.m.; n = 3).
MCUR1 is an essential component of mitochondrial Ca2+ uptake that regulates cellular metabolism (2012)
figf5

(a) AMP/ATP ratios in stable HeLa cell lines stably expressing negative shRNA (Neg shRNA), MCUR1 shRNA (clone shHe2) or shHe2 with MCUR1 re-expressed. **P0.01 (mean±s.e.m.; n = 3). (b) O2 consumption rates (OCR) in stable HeLa cells expressing irrelevant shRNA, clone shHe2, and clone shHe2 re-expressing MCUR1, exposed sequentially to oligomycin (i), FCCP (ii) and rotenone plus myzothiazol (iii). (c) Basal and maximal O2 consumption rates in cells as described in b. *P0.05 (mean±s.e.m.; n = 3). (d) Western blot of phosphorylated and total AMPK (top) and densitometric analysis (bottom) in stable HeLa lines expressing negative shRNA or MCUR1 shRNA (clone shHe2) and clone shHe2 re-expressing MCUR1. *P0.05, **P0.01 (mean±s.e.m.; n = 3). (e) Western blot of LC3 or tubulin in stable HeLa lines expressing negative shRNA or MCUR1 shRNA (clone shHe2) and clone shHe2 re-expressing MCUR1 (top) and quantification of LC3-II/(LC3-I + LC3-II) (bottom) expressed as fold increase over levels in cells expressing irrelevant shRNA. *P0.05, **P0.01 (mean±s.e.m.; n = 3). (f,g), The same as in d and e. Activation of AMPK (f) and autophagy (**P0.001; mean±s.e.m.; n = 3) (g) in the absence and presence of InsP3R inhibitor xestospongin B (XeB). *P0.05, **P0.001, NS, not significant (mean±s.e.m.; n = 3). Uncropped images of blots/gels are shown in Supplementary Fig. S6.
Systematic analysis of IL-17 receptor signalosome reveals a robust regulatory feedback loop (2020)
Figure 1

Figure 1. Kinases TBK1 and IKKε are major components of IL-17RSC

A. Schematic representation of IL-17RSC isolation and analysis. Cells are stimulated with recombinant Strep-Flag-IL-17 (SF-IL-17) (1), which leads to the crosslinking of its two receptor subunits and recruitment of cytoplasmic molecules (2). The whole complex is isolated upon cell lysis via tandem affinity purification of the ligand and analyzed by MS (3).

B. ST2 cells were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. As a control, cells were first solubilized and SF-IL-17 was added only post lysis. Number of identified peptides (unique + razor) and iBAQ intensities for each protein in five independent experiments are shown.

C. The stoichiometry of IL-17RSC calculated as the ratio between iBAQ intensities of individual IL-17RSC components to iBAQ intensity of IL-17RC. The recruitment of kinases TBK1 and IKKε as compared to related kinases IKKα and IKKβ is significantly enhanced. Mean from five performed MS experiments is shown, the statistical significance was determined using unpaired two‐tailed Student's t‐test.

D. ST2 cells were stimulated with IL-17 (500 ng/ml), TNF (50 ng/ml), or IL-1α (50 ng/ml) for indicated time points and activation of signaling pathways was analyzed by immunoblotting. A representative of two independent experiments is shown.
Systematic analysis of IL-17 receptor signalosome reveals a robust regulatory feedback loop (2020)
Figure 2

Figure 2. TBK1 and IKKε function in redundant manner as inhibitors of IL-17-induced signaling responses

A-B. ST2 cells were either left untreated (A) or pretreated with TBK1/IKKε inhibitor MRT67307 (2 µM) for 30 minutes (B), followed by stimulation with IL-17 (500 ng/ml) for 2 hours. Transcription response induced by IL-17 stimulation as compared to unstimulated cells was analyzed by RNA sequencing. In red are transcripts considered to be significantly changed (log2 fold change > 1 or < -1, - log10 Benjamini-Hochberg adjusted p-value > 2, based on the analysis of three independent experiments; tringle is used for transcripts with - log10 adjusted p-value > 100). Names of several significantly upregulated transcripts are indicated.

C. The Venn diagram representing the number of significantly changed transcripts upon IL-17 stimulation detected in (A) and (B).

D-E. Comparison of IL-17-induced transcriptional response in presence or absence of TBK1/IKKε inhibitor. (D) Transcripts that are significantly changed in both conditions. (E) Transcripts that pass significance threshold for induction only in presence of TBK1/IKKε inhibitor. Dashed lines indicate significantly upregulated transcripts (log2 fold change > 1); red lines separates transcripts that are more upregulated upon IL-17 stimulation in the presence of TBK1/IKKε inhibitor as compared to IL-17 alone.

F. ST2 cells pretreated or not with TBK1/IKKε inhibitor MRT67307 (2 µM) were left unstimulated or stimulated with IL-17 (500 ng/ml) for 2 hours and induction of mRNA for selected genes was analyzed by real time PCR. Mean + SEM from five independent experiments is shown, statistical significance was determined using unpaired two‐tailed Student's t‐test.

G. ST2 cells were pretreated or not with TBK1/IKKε inhibitor MRT67307 (2 µM), stimulated with IL17 (500 ng/ml) for indicated time points and analyzed by immunoblotting.

H. ST2 wild type, TBK1 KO, IKKε KO, or cells lacking both kinases (DKO) were stimulated with IL-17 (500 ng/ml) as indicated and analyzed by immunoblotting.

I. TBK1/IKKε DKO cells reconstituted with either wild type or kinase dead mutant (K38A) version of both kinases were stimulated with IL-17 (500 ng/ml) as indicated and analyzed by immunoblotting.

Data information: Immunoblot results are representative of four (G) or three (H, I) independent experiments.