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: International journal of biological sciences

Article Title: The RNF26/CBX7 axis modulates the TNF pathway to promote cell proliferation and regulate sensitivity to TKIs in ccRCC.

doi: 10.7150/ijbs.69325

Figure Lengend Snippet: Figure 6. RNF26 promotes the degradation of CBX7 in renal cancer cells. A, A498 and 786-O cells were infected with indicated shRNAs for 72 h. After treated with or without MG132 for another 8 h, cells were harvested for Western blot analysis. B, A498 and 786-O cells were transfected with indicated plasmids for 24 h. After treated with or without MG132 for another 8 h, cells were harvested for Western blot analysis. C, A498 and 786-O cells were transfected with indicated plasmids for 24 h. Cells were harvested for Western blot analysis. D, 786-O cells were infected with the indicated shRNAs. After 72 h, cells were treated with cycloheximide (CHX), and cells were collected for Western blot analysis at different time points. E, 786-O cells were transfected with the indicated plasmids. After 24 h, cells were treated with CHX, and cells were collected for Western blot analysis at different time points. F, 786-O cells were transfected with the indicated plasmids. After 24 h, cells were collected for Western blot after treatment with MG132 for 8 h. G, 786-O cells were infected with the indicated shRNAs. After 72 h, cells were collected for Western blot after treatment with MG132 for 8 h.

Article Snippet: MG132 (#S2619), IKK-16 (#S2882), JSH-23 (#S7351) and cycloheximide (CHX) (#S7418) were purchased from Selleckchem.

Techniques: Infection, Western Blot, Transfection

Journal: Scientific reports

Article Title: The N-Acetyl Phenylalanine Glucosamine Derivative Attenuates the Inflammatory/Catabolic Environment in a Chondrocyte-Synoviocyte Co-Culture System.

doi: 10.1038/s41598-019-49188-9

Figure Lengend Snippet: Figure 5. IL-1β increases IKKα nuclear translocation and phosphorylation of serine 10 on histone H3, while NAPA attenuates these signalling events. (A) Overlapping signals of nuclear counterstaining (DAPI) and IKKα detected via an Alexa Fluor 555 secondary antibody: the colocalized signals indicate that NAPA addition is effective in reducing nuclear translocation of IKKα. (B) Upper pictures: left, western blot of anti- phosphorylated serine 10 of histone H3, along with GAPDH as a loading control and right: densitometric analysis of the signal showing the different pattern of H3pSer10 accumulation in CTR (circle), IL-1β (square) or IL-1β + NAPA (triangle) conditions. Lower images: specificity of the signal obtained with the anti-H3 phosphorylated serine 10: 20x field pictures of chondrocytes grown on coverslips in the bottom of wells at time 0 in control (upper row) or IL-1β stimulated conditions (lower row): Green: IKKα detected with an Alexa Fluor 488 anti-rabbit antibody; red: H3pSer10 signal detected with an Alexa Fluor 555 anti-mouse antibody; blue: nuclear DNA stained with Hoechst 33342 and merged images.

Article Snippet: IKKα staining was performed with 5 μg/ml rabbit anti-IKKα antibody (BOSTER Biological Technology, Freemont, CA, USA), while the extent of phosphorylation of serine 10 of histone H3 was evaluated with 5 μg/ml anti-phospho-histone H3, (Ser10), (mouse monoclonal, Upstate–Millipore) overnight at 4 °C.

Techniques: Translocation Assay, Phospho-proteomics, Western Blot, Control, Staining