human ikkβ  (Addgene inc)

Journal: Life Science Alliance

Article Title: TLR-induced STK25 activation promotes IRF5-mediated inflammation

doi: 10.26508/lsa.202503343

Figure Lengend Snippet: (A) Dose-dependent phosphorylation of a biotinylated C-terminal construct of IRF5 (residues 222–467, 3 μM) by candidate kinases in an in vitro scintillation proximity assay ( N = 4 biological replicates). (B, C, D, E) Phosphorylation of full-length IRF5 by IKKβ (B, C) or STK25 (D, E) in an in vitro luminescent kinase assay. (B) Dose-dependent phosphorylation of IRF5 (500 ng) by IKKβ. (C) Dose-dependent phosphorylation of IRF5 by a fixed amount of IKKβ (100 ng). (D) Dose-dependent phosphorylation of IRF5 (500 ng) by STK25. (E) Dose-dependent phosphorylation of IRF5 by a fixed amount of STK25 (350 ng). (F) Phos-tag immunoblot analysis of the kinetics of STK25- and IKKβ-mediated phosphorylation of full-length IRF5 in vitro. Blots were probed with an antibody against total IRF5. p-IRF5, phosphorylated IRF5. (G) Immunoblot analysis of IRF5 phosphorylation at threonine residues following incubation with STK25 in an in vitro kinase assay for 1 h at RT. Blots were probed with antibodies against total p-Thr and IRF5. Representative of three independent experiments. p-Thr, phosphorylated threonine. (H) Mass spectrometry-based identification of Thr265 as an STK25-dependent IRF5 phosphorylation site. In vitro kinase reactions with IRF5 and STK25 were incubated for 1 h at RT, subjected to SDS–PAGE, and the gel was stained with Coomassie blue. Protein bands were excised, destained, and digested with trypsin. Representative of three independent experiments. (I) Conservation of Thr265 across multiple human isoforms of IRF5. (J) Conservation of Thr265 in IRF5 protein sequences from multiple species. (K) WT-IRF5 and IRF5-T265A were generated via an in vitro cell-free protein expression system and evaluated by immunoblot analysis. Blots were probed with an antibody against total IRF5. (L) Phosphorylation of WT-IRF5 and IRF5-T265A by STK25 in an in vitro luminescent kinase assay ( N = 3 biological replicates). (M) Immunoblot analysis of WT-IRF5 and IRF5-T265A phosphorylation at threonine residues following incubation with STK25 in an in vitro kinase assay for 1 h at RT. Blots were probed with antibodies against total p-Thr and IRF5. (N) Densitometric analysis of WT-IRF5 and IRF5-T265A phosphorylation at threonine residues after normalization to the expression of total IRF5 ( N = 3 biological replicates). * P < 0.05, ** P < 0.01. Data represent mean ± SEM.

Article Snippet: Recombinant human IKKβ (I03-10BG-10; Signalchem).

Techniques: Phospho-proteomics, Construct, In Vitro, Scintillation Proximity Assay, Kinase Assay, Western Blot, Incubation, Mass Spectrometry, SDS Page, Staining, Generated, Expressing

Journal: Life Science Alliance

Article Title: TLR-induced STK25 activation promotes IRF5-mediated inflammation

doi: 10.26508/lsa.202503343

Figure Lengend Snippet: (A) Immunoblot analysis of STK25 autophosphorylation at Thr174 in THP-1 cells stimulated with LPS for 0.5, 1, 2, 4, 6, or 24 h. Blots were probed with antibodies against p-STK25 (T174), STK25, and GAPDH. (B) Densitometric analysis of p-STK25 (T174) protein levels after normalization to total STK25 protein levels and the expression of GAPDH ( N = 2 independent experiments). (C) Immunoblot analysis of STK25 autophosphorylation at Thr174 in Ramos B cells stimulated with CpG-B for 0.5, 1, 2, 4, 6, or 24 h. Blots were probed with antibodies against p-STK25 (T174), STK25, and GAPDH. (D) Densitometric analysis of p-STK25 (T174) protein levels after normalization to total STK25 protein levels and the expression of GAPDH ( N = 2 independent experiments). (E, F, G, H, I) Flow cytometry analysis of WT and KO splenocyte viability for uncultured ( N = 8–13 biological replicates per genotype) (E), untreated ( N = 4–5 biological replicates per genotype) (F), R848-stimulated ( N = 6 biological replicates per genotype) (G), CpG-B-stimulated ( N = 6 biological replicates per genotype) (H), and LPS-stimulated ( N = 6 biological replicates per genotype) (I) cells used for 24 h cytokine release assays. (J) Immunoblot analysis of blot overlay assays conducted with full-length IRF5 (100 ng) and varying amounts (50–200 ng) of full-length IKKβ, Lyn, or STK25. Blots were probed with an antibody against IRF5.

Article Snippet: Recombinant human IKKβ (I03-10BG-10; Signalchem).

Techniques: Western Blot, Expressing, Flow Cytometry

Journal: Life Science Alliance

Article Title: TLR-induced STK25 activation promotes IRF5-mediated inflammation

doi: 10.26508/lsa.202503343

Figure Lengend Snippet: (A) Immunoblot analysis of STK25 autophosphorylation at Thr174 in PBMCs from healthy donors and patients with systemic lupus erythematosus. Blots were probed with antibodies against p-STK25 (T174), STK25, and β-actin. (B) Densitometric analysis of total STK25 protein levels after normalization to the expression of β-actin ( N = 8–9 samples per condition). (C) Densitometric analysis of p-STK25 (T174) protein levels after normalization to total STK25 protein levels and the expression of β-actin ( N = 8–9 samples per condition). Data represent mean ± SEM. (D) Proposed model for STK25 as an IRF5 kinase downstream of TLR7/8/9 signaling (pathway highlighted by the red dashed arrows). The activation of TLR7/8 by ssRNA or TLR9 by CpG-B DNA induces the autophosphorylation of STK25 at Thr174 and stimulates the formation of the Myddosome, a signaling complex comprised of MyD88, IRAK1/4, TRAF6, and IRF5. The activation of IRAK1 by IRAK4 leads to the recruitment of TRAF6. The ubiquitination (colored circles) of TRAF6 provides a binding site for TAB2/3, thereby facilitating the activation of TAK1. NEMO, a component of the IKK complex, interacts with ubiquitinated TRAF6 to promote the activation of IKKβ by TAK1. IKKβ-mediated phosphorylation of IκBα induces its ubiquitination and degradation by the 26S proteasome. The inhibition of IκBα permits the activation and nuclear translocation of the RelA and p50 subunits of NF-κB. In the nucleus, NF-κB regulates the expression of pro-inflammatory cytokines. In the alternate arm of the pathway, IRF5 undergoes TRAF6-catalyzed ubiquitination in addition to IKKβ- and/or STK25-dependent phosphorylation at Ser446 and Thr265, respectively. The activation of IRF5 also involves an interaction with the adaptor protein, TASL, which binds to SLC15A4. Whereas IKKβ is required for TASL-mediated activation of IRF5, it is unknown if STK25 modulates the TASL-dependent pathway. Altogether, these modifications induce IRF5 dimerization and nuclear translocation. In the nucleus, transcriptionally active IRF5 modulates the expression of pro-inflammatory cytokines and type I interferons. Created with BioRender.com .

Article Snippet: Recombinant human IKKβ (I03-10BG-10; Signalchem).

Techniques: Western Blot, Expressing, Activation Assay, Ubiquitin Proteomics, Binding Assay, Phospho-proteomics, Inhibition, Translocation Assay

Journal: Frontiers in Immunology

Article Title: Fbxo16 mediates degradation of NF-κB p65 subunit and inhibits inflammatory response in dendritic cells

doi: 10.3389/fimmu.2025.1524110

Figure Lengend Snippet: Fbxo16 forms a CRL1 complex, binds to p65 and negatively regulates NF-κB signaling. (A) Interaction of Fbxo16 and Cullins in whole cell lysates of HEK293T cells, transfected with c-Myc-Fbxo16, then immunoprecipitated with anti-Flag, and immunoblotted with anti-Cullin 1, Cullin 2 or Cullin 3. (B) Interaction of Fbxo16 and Skp1 in whole cell lysates of HEK293T cells, transfected with c-Myc-Fbxo16, then immunoprecipitated with anti-c-Myc, and immunoblotted with anti-Skp1. (C) Interaction of Fbxo16 and p65 in whole cell lysates of HEK293T cells, transfected with a Flag-p65 or p50 along without or with c-Myc-Fbxo16, then immunoprecipitated with anti-c-Myc, and immunoblotted with anti-p65 or p50. (D) Interaction of Fbxo16 and PDLIM2 in whole cell lysates of HEK293T cells, transfected with a Flag-PDLIM2 along without or with c-Myc-Fbxo16, then immunoprecipitated with anti-c-Myc, and immunoblotted with anti-Flag. (E) Luciferase assay in MEFs transfected with ELAM−1 luciferase reporter and pRL-Null renilla constructs along with increasing amounts (wedge) of Fbxo16, then left untreated or stimulated with LPS for 5 hr. (F) Luciferase assay in HEK293T cells transfected with ELAM−1 luciferase reporter and pRL-Null renilla constructs without or with MyD88, IKKβ, TRAF6 or p65 along with increasing amounts (wedge) of Fbxo16. Data are representative of three (A, B) , four (C, D) or three ( E, F ; means ± SD, **P<0.01) independent experiments.

Article Snippet: Expression plasmids for Flag-tagged murine p50 (#20018), murine TRAF6 (#21624), murine MyD88 (#13093) and human IKKβ (#23298) were purchased from Addgene.

Techniques: Transfection, Immunoprecipitation, Luciferase, Construct