Journal: eLife

Article Title: Dual-specific autophosphorylation of kinase IKK2 enables phosphorylation of substrate IκBα through a phosphoenzyme intermediate

doi: 10.7554/eLife.98009

Figure Lengend Snippet: ( A ) A schematic of the IKK-complex (pre- and post-stimulation) showing binding of NEMO helping activation of IKK as well as channelizing its recognition of substrate IκBα. The mechanism of specific phosphorylation at Ser32 and Ser36 of IκBα is unclear ( B ) Domain organization of IKK2 based on the X-ray structures highlighting its functional kinase domain (KD), ubiquitin-like domain (ULD), scaffold dimerization domain (SDD), and NEMO-binding domain. Serine residues in the activation loop - substitution of which to glutamate renders IKK2 constitutively active, and those in the SRR region known to be phosphorylated are marked. Tyrosine residues in the activation loop and the conserved ATP-interacting Lys44 are also marked. ( C ) In vitro kinase assay showing autophosphorylation of wild-type full-length IKK2 (FL IKK2WT) upon incubation with γ 32 P radiolabeled ATP for different time periods. ( D ) Similar in vitro kinase assays performed to assess the effect of NEMO on autophosphorylation of FL IKK2WT (left panel), and the effect of NEMO and IκBα on autophosphorylation and substrate phosphorylation (right panel) activities of FL IKK2WT. ( E ) In vitro kinase assay (schematic depicted in ) showing the effect of different concentrations of the Inhibitor VII on FL IKK2WT autophosphorylation and IκBα substrate phosphorylation (this assay was performed twice). ( F ) Kinase assay with radiolabeled ATP displaying auto- and substrate-phosphorylation of full-length and deletion constructs of the constitutively active form of IKK2 harboring phosphomimetic Ser177Glu and Ser181Glu substitutions. Figure 1—source data 1. Original unedited autoradiograph and Coomassie-stained gel files used in . Figure 1—source data 2. Original autoradiographs and Coomassie-stained gel files used in with sample labels.

Article Snippet: Chemical compound, drug , IKK Inhibitor VII , Calbiochem , CAS 873225-46-8 , IKK inhibitor.

Techniques: Binding Assay, Activation Assay, Phospho-proteomics, Functional Assay, Ubiquitin Proteomics, In Vitro, Kinase Assay, Incubation, Construct, Autoradiography, Staining

Journal: mBio

Article Title: Cytomegalovirus-induced peroxynitrite promotes virus entry and contributes to pathogenesis in a murine model of infection

doi: 10.1128/mbio.03152-23

Figure Lengend Snippet: Peroxynitrite is essential for HCMV infection in PMA-differentiated THP1 cells in vitro . ( A ) Peroxynitrite production in PMA-differentiated THP-1 cells treated with peroxynitrite scavengers before cell-free TB40-BAC4 infection (MOI 25). ( B ) HCMV was pre-incubated with Cytotect or seronegative IgG (each at 40 µg/mL) before peroxynitrite measurement. ( C ) Cells were pre-treated with neutralizing antibody against TLR2 or IgG control (200 µg/mL) before the addition of HCMV and measurement of peroxynitrite generation. ( D ) Percentage of HCMV infected (GFP+) THP-1 cells 72 hpi, following pre-treatment with inhibitors ( n = 4–7). Data plotted as mean ± SEM. Statistical analysis using the Kruskal-Wallis test with Dunn’s multiple comparisons (**** P < 0.0001; *** P < 0.0005; * P < 0.01, and ns, not significant). The gating strategy and representative flow cytometry plots are shown in ( E ). Only live cells were selected for analysis. ( F ) Peroxynitrite production in differentiated THP-1 cells treated with serotonin before cell-free TB40-BAC4-GFP infection (MOI 25). ( G ) Representative flow cytometry plots of HCMV infected (GFP+) THP-1 cells pre-treated with serotonin, MnTBAP, or vehicle. ( A-B & E ) Fluorescence intensity was measured every 10 s for 180 min. Average fluorescence was plotted ( n = 2). Cells were pre-treated with FeTPPS (25 µM), FeTMpYP (25 µM), NAC (10 mM), Serotonin (250–500μM), MnTBAP (50 µM), or vehicle. Data are representative of at least two separate experiments.

Article Snippet: For assays targeting NF-κB or TLR2 signaling, IKK-16 (NF-κB inhibitor; IKK inhibitor VII, 1 nM; Cambridge Bioscience) or neutralizing antibodies to TLR2 (200 μg/mL; Invivogen) were added 1 h before the addition of HCMV ( ).

Techniques: Infection, In Vitro, Incubation, Control, Flow Cytometry, Fluorescence