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: Molecular cancer therapeutics

Article Title: PIM kinase inhibitors block the growth of primary T-cell acute lymphoblastic leukemia: Resistance pathways identified by network modeling analysis

doi: 10.1158/1535-7163.MCT-20-0160

Figure Lengend Snippet: (A) HEK-293T-NF-κB-luc cells were incubated with conditioned media from HSB-2 naïve or AZDR1 and LGBR2 cells with or without IKK16 (2 μM) for 6h. (B & C) HEK-293T-NF-κB-luc cells were incubated with media containing TNF-α (20 ng/mL) alone or in combination with IKK16 (2 μM) or CM from HSB-2 LGBR2 or AZDR1 with or without IKK16 (2 μM) for 6h. NF-κB mediated luciferase activity was measured using One-Glo Luciferase Assay System. Cell lysates were Western blotted with the specified antibodies. LE: long exposure. (D) Cell viability of HSB-2 naïve and AZDR1 cells treated with indicated concentrations of AZD alone and/or in combination with IKK16 for 72h. (E) T-ALL PDX cells were incubated with the indicated concentrations of LGB321 (LGB) alone or in combination with IKK16 for 72h and then the percentage of viable cells was quantified by the ATPlite assay. For (D and E), the growth of DMSO control cells was considered 100% and percent cell growth after individual treatment is reported relative to the DMSO. The data shown are the average +/− S.D. of three independent experiments.

Article Snippet: LGB-321 (Cat # A14420), AZD1208 (Cat # A13203), and IKK16 (# A12836) were purchased from Adooq bioscience.

Techniques: Incubation, Luciferase, Activity Assay, Western Blot