Journal: Biochemical and biophysical research communications

Article Title: IKK regulates the deubiquitinase CYLD at the postsynaptic density.

doi: 10.1016/j.bbrc.2014.06.019

Figure Lengend Snippet: Fig. 2. IKK phosphorylates CYLD at S-418 in vitro. (A) PSD fractions were incubated under different conditions designed to manipulate IKK activity, followed by Western immunoblotting with an antibody specific to CYLD phosphorylated at S- 418 (p-CYLD) (top panels) or an antibody to CYLD (bottom panels). Addition of ATP induced phosphorylation of CYLD at S-418, but inclusion of the IKK inhibitors, IKK16 or tatNEMO reduced CYLD phosphorylation in a dose-dependent manner. (B) Purified CYLD and purified IKKb were incubated in the presence or the absence of ATP, followed by Western immunoblotting as described above. Addition of ATP induced phosphorylation of CYLD at S-418. Two experiments yielded similar results.

Article Snippet: IKK16, N-(4-Pyrrolidin-1-yl-piperidin-1-yl)-[4-(4-benzo[b]thiophen-2-yl-pyrimidin2-ylamino)phenyl]carboxamide hydrochloride, is from Tocris (Ellisville, CO).

Techniques: In Vitro, Incubation, Activity Assay, Western Blot, Phospho-proteomics

Journal: Biochemical and biophysical research communications

Article Title: IKK regulates the deubiquitinase CYLD at the postsynaptic density.

doi: 10.1016/j.bbrc.2014.06.019

Figure Lengend Snippet: Fig. 3. The phosphorylation state of CYLD at the PSD of hippocampal neurons is controlled by IKK under basal conditions. Hippocampal cultures were pre- incubated with or without the IKK inhibitor IKK16 for 20 min, followed by treatment with either NMDA or control medium for 2 min before fixation and immunogold labeling with an antibody specific for CYLD phosphorylated on S-418 (p-CYLD). (A) Electron micrographs of the synaptic region showing immunogold labeling for p-CYLD, seen as dark grains of heterogeneous sizes. On the right panel is shown the zone for measuring amount of labeling at the PSD complex, delineated by two perpendicular lines to the postsynaptic membrane and a parallel dashed line at 120 nm distance from the postsynaptic membrane. NMDA treatment promoted an increase in p-CYLD labels at the PSD. (B and C) The histograms depict the frequency of PSDs either with no label (left) or with labels of varying intensities (right). Immunolabeling intensities were binned into groups from 5 to 65. Under basal conditions (B), the IKK inhibitor IKK16 (gray bars) reduced the labeling for phosphorylated CYLD at the PSD. However, preincubation with the inhibitor had no significant effect on labeling for phosphorylated CYLD following NMDA treatment (C). The histograms represent one experiment out of a total of three experiments with similar results.

Article Snippet: IKK16, N-(4-Pyrrolidin-1-yl-piperidin-1-yl)-[4-(4-benzo[b]thiophen-2-yl-pyrimidin2-ylamino)phenyl]carboxamide hydrochloride, is from Tocris (Ellisville, CO).

Techniques: Phospho-proteomics, Incubation, Control, Labeling, Membrane, Immunolabeling

Journal: Biochemical and biophysical research communications

Article Title: IKK regulates the deubiquitinase CYLD at the postsynaptic density.

doi: 10.1016/j.bbrc.2014.06.019

Figure Lengend Snippet: Fig. 4. IKK regulates DUB activity targeting K63-linked polyubiquitins. (A) Purified CYLD was pre-incubated with purified IKKb in the presence or absence of ATP, followed by incubation with tetrameric K63-linked polyubiquitin chains (Ub4). IKKb was omitted in additional controls (right column). The upper two panels are Western immunoblots with antibodies specific for CYLD phosphorylated at S-418 (p-CYLD) and for CYLD respectively, while the bottom panels shows the lower portions of coomassie blue stained gels containing Ub4 and degradation products. Addition of both IKKb and ATP promotes CYLD phosphorylation (upper panels), with a concomitant increase in the rate of cleavage of Ub4 (bottom panels). Two experiments yielded similar results. (B) PSD fractions were incubated under different conditions designed to manipulate IKK activity, followed by incubation with tetrameric K63-linked polyubiquitin chains (Ub4). CYLD phosphorylation status (top panels) in comparison to total CYLD levels (middle panels) and DUB activity (bottom panels) were monitored as described above. Inclusion of ATP promoted CYLD phosphorylation, and an increase in the rate of Ub4 breakdown. Addition of IKK16 prevented both CYLD phosphorylation and ATP-dependent upregulation of Ub4 breakdown (bottom panels). Three independent experiments yielded similar results.

Article Snippet: IKK16, N-(4-Pyrrolidin-1-yl-piperidin-1-yl)-[4-(4-benzo[b]thiophen-2-yl-pyrimidin2-ylamino)phenyl]carboxamide hydrochloride, is from Tocris (Ellisville, CO).

Techniques: Activity Assay, Incubation, Western Blot, Staining, Phospho-proteomics, Comparison

Journal: Journal of Clinical Investigation

Article Title: Interplay of IKK/NF-κB signaling in macrophages and myofibers promotes muscle degeneration in Duchenne muscular dystrophy

doi: 10.1172/jci30556

Figure Lengend Snippet: Figure 1 NF-κB activity in dystrophic muscle is localized to both muscle and immune cells. (A–C) Muscle nuclear extracts from 5-week-old WT C57BL/10 and 3- and 5-week-old mdx mice (A), 5-week-old WT and mdx mice (B), or 7-week-old WT, mdx, and DKO mice (C) were used in EMSA for NF-κB and Oct-1. Supershift assays were performed on mdx muscle extracts using antibodies against p65 and p50. Arrowheads denote shifted subunits. (D) IKK assays performed with IκBα WT and mutant (double serine to threonine [SS/TT]) or p65 WT and mutant (serine to alanine [S/A]) substrates using gastrocnemius muscle lysates from 7-week-old WT or mdx mice. Immunoprecipitates were probed for IKKγ as a loading control. Western blots are shown for p-IKK, p-IκBα, IκBα, p-p65, and p65. GST, glutathione-S-transferase. (E) Gastrocnemius muscles from 7-week-old WT or mdx mice were immunostained for p-p65. Scale bars: 50 μm. Black arrowheads denote immune cells, and blue arrowheads indicate regenerating fibers. (F) Muscles from either 4- or 7-week-old mdx mice were double stained with p-p65 (green) and CD68 (red) or p-p65 and E-MyHC (red), respectively. Scale bars: 20 μm. (G) H&E staining and p-p65 immunohistochemistry were performed on muscle biopsies from healthy controls and age-matched DMD patients (n = 4). Scale bar: 50 μm.

Article Snippet: Muscle extracts for Western blot analysis were prepared as previously described (52) and probed using antibodies against p-p65, p-IκB, pIKK (1:500; Cell Signaling Technology), IκBα (1:500; Santa Cruz Biotechnology Inc.), IKKα (1:500; Imgenex), IKKβ 900 The Journal of Clinical Investigation http://www.jci.org Volume 117 Number 4 April 2007 (1:500; Cell Signaling Technology), IKKγ (1:500; Santa Cruz Biotechnology Inc.) and p65 (1:10,000; Rockland).

Techniques: Activity Assay, Mutagenesis, Control, Western Blot, Muscles, Staining, Immunohistochemistry

Journal: Journal of Clinical Investigation

Article Title: Interplay of IKK/NF-κB signaling in macrophages and myofibers promotes muscle degeneration in Duchenne muscular dystrophy

doi: 10.1172/jci30556

Figure Lengend Snippet: Figure 6 IKKβ deletion in muscle cells promotes regeneration. Muscles harvested from 4-week- (A–F) or 12-week-old (F only) mdx;IKKβF/F and mdx;IKKβF/F;MLC-Cre mice were used for protein analysis (A and C) or histology (B and D). (A) Western blots probing for IKK subunits. (B) Muscles were immunostained for p-p65 expression. (C) Western blots probing for p-p65, p65, and IκBα. (D) H&E staining of mdx;IKKβF/F and mdx;IKKβF/F;MLC-Cre muscles. (E) Muscles used in D were stained for quantitation of E-MyHC–positive stained fibers or stained with H&E for counting of CLN. *P < 0.05. Scale bars: 15 μm (B) and 50 μm (D). Graphs are plotted as mean ± SEM. (F) Mean fiber distribution in mdx;IKKβF/F and mdx;IKKβF/F;MLC-Cre mice was determined from a minimum of 3,000 fibers from randomly chosen fields, obtained from multiple muscle sections from a minimum of 4 mice per group.

Article Snippet: Muscle extracts for Western blot analysis were prepared as previously described (52) and probed using antibodies against p-p65, p-IκB, pIKK (1:500; Cell Signaling Technology), IκBα (1:500; Santa Cruz Biotechnology Inc.), IKKα (1:500; Imgenex), IKKβ 900 The Journal of Clinical Investigation http://www.jci.org Volume 117 Number 4 April 2007 (1:500; Cell Signaling Technology), IKKγ (1:500; Santa Cruz Biotechnology Inc.) and p65 (1:10,000; Rockland).

Techniques: Muscles, Western Blot, Expressing, Staining, Quantitation Assay

Journal: Journal of Clinical Investigation

Article Title: Interplay of IKK/NF-κB signaling in macrophages and myofibers promotes muscle degeneration in Duchenne muscular dystrophy

doi: 10.1172/jci30556

Figure Lengend Snippet: Figure 8 Pharmacological inhibition of IKK rescues the histopathology and function of dystrophic muscle. (A) Amino acid sequence of WT or mutant (mut) NBD peptides. Underlined amino acids indicate changes from WT to mutant forms. (B) Soleus muscles from WT or mutant NBD–treated mice were stained with F4/80, and macrophages were quantitated. Scale bar: 300 μm. (C) Gastrocnemius muscles harvested from mdx mice treated for 4 weeks were sectioned and stained with either p-p65 (C) or H&E (E). Scale bars: 20 μm (C and E). (D) Lysates from mice were used for Western blots probing for p-p65, p65, and IκBα. (F) RNA was isolated from similar muscles as used for D including C57BL/10 and mdx controls, and real-time PCR was performed for lysozyme (n = 4). (G) Regeneration potential was measured by quantitating fibers with centronucleation and positive E-MyHC staining from mdx mice treated with saline or NBD peptides. (H) Force generation assessed by measur- ing active developed force comparing diaphragm muscles from mice treated with either WT or mutant NBD peptide for 4 weeks. Quantitative data are plotted as mean ± SEM from 3 independent experiments. *P < 0.05.

Article Snippet: Muscle extracts for Western blot analysis were prepared as previously described (52) and probed using antibodies against p-p65, p-IκB, pIKK (1:500; Cell Signaling Technology), IκBα (1:500; Santa Cruz Biotechnology Inc.), IKKα (1:500; Imgenex), IKKβ 900 The Journal of Clinical Investigation http://www.jci.org Volume 117 Number 4 April 2007 (1:500; Cell Signaling Technology), IKKγ (1:500; Santa Cruz Biotechnology Inc.) and p65 (1:10,000; Rockland).

Techniques: Inhibition, Histopathology, Sequencing, Mutagenesis, Muscles, Staining, Western Blot, Isolation, Real-time Polymerase Chain Reaction, Saline

Journal: Bone & Joint Research

Article Title: Therapeutic potential of IκB kinase epsilon inhibition in preventing meniscal degeneration of early osteoarthritis

doi: 10.1302/2046-3758.1411.BJR-2024-0518.R2

Figure Lengend Snippet: IκB kinase (IKKε) overexpression activated nuclear factor-κB (NF-κB) signalling in osteoarthritis (OA) human meniscal cells in the presence of interleukin-1β (IL-1β), and this effect was reversed by amlexanox and BAY-985 treatment. a) Western blot analysis and b) quantification of p-IκBα, IκBα, p-p65, and p65 levels in OA human meniscal cells after IKKε overexpression and treatment with amlexanox (100 μM) or BAY-985 (10 μM), either unstimulated or 20 minutes after IL-1β (0.1 ng/ml) stimulation. Green fluorescent protein adenoviral vector (Ad-GFP) was used as a vector control. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. All p-values in this figure were calculated using Tukey-Kramer test or Steel-Dwass test. Symbols represent individual samples; thick horizontal lines with whiskers show the mean (SD). *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: Knee joint sections were stained with a primary antibody against IKKε (SC376114; Santa Cruz Biotechnology), p-IκBα (NB100-81987; Novus Biologicals, USA), IL-6 (product no. 12912; Cell Signaling Technology), or MMP13 (ab39012; Abcam) at room temperature for one hour, then incubated with Alexa Fluor-conjugated secondary antibodies (Thermo Fisher Scientific).

Techniques: Over Expression, Western Blot, Plasmid Preparation, Control

Journal: Bone & Joint Research

Article Title: Therapeutic potential of IκB kinase epsilon inhibition in preventing meniscal degeneration of early osteoarthritis

doi: 10.1302/2046-3758.1411.BJR-2024-0518.R2

Figure Lengend Snippet: IκB kinase (IKKε) knockout attenuates the progression of meniscal degeneration in an early osteoarthritis (OA) mouse model. a) Left panel: representative images of wild-type (WT) and IKKε knockout (KO) mice showing their growth and development, as well as changes in body weight at the indicated ages. Middle panel: representative Safranin O–fast green staining images of the menisci of untreated WT and IKKε-KO mice at 12 weeks of age. Right panel: quantification of pathological changes in meniscal degeneration in each group. Values represent the mean (SD) (n = 5 mice per group). b) Representative Safranin O–fast green staining images of the menisci of WT and IKKε-KO mice four weeks after destabilization of the medial meniscus (DMM) + medial collateral ligament (MCL) transection surgery (top). Quantification of the severity of meniscal degeneration and articular cartilage histopathological changes in the indicated groups (bottom) (n = 7 mice per group). c) Representative immunofluorescence images (left) and quantification (right) of p-IκBα, interleukin-6 (IL-6), and matrix metalloproteinase-13 (MMP-13) expression in the menisci of WT and IKKε-KO mice four weeks after DMM + MCL transection surgery. Right panels show higher-magnification views of the boxed areas in the left panels (bar = 100 μm). All p-values in this figure were calculated using independent-samples t -test or Mann-Whitney U test. Symbols represent individual samples; thick horizontal lines with whiskers show the mean (SD). *p < 0.05; **p < 0.01.

Article Snippet: Knee joint sections were stained with a primary antibody against IKKε (SC376114; Santa Cruz Biotechnology), p-IκBα (NB100-81987; Novus Biologicals, USA), IL-6 (product no. 12912; Cell Signaling Technology), or MMP13 (ab39012; Abcam) at room temperature for one hour, then incubated with Alexa Fluor-conjugated secondary antibodies (Thermo Fisher Scientific).

Techniques: Knock-Out, Staining, Immunofluorescence, Expressing, MANN-WHITNEY