Journal: Cells

Article Title: IL-3-Induced Immediate Expression of c- fos and c- jun Is Modulated by the IKK2-JNK Axis

doi: 10.3390/cells11091451

Figure Lengend Snippet: Effects of IKK1 and/or IKK2 KO on IL-3-induced expression of IEGs. ( A ) IKK1 and IKK2 protein levels in Ba/F3 parental cells (WT) and CRISPR/Cas9-mediated IKK1 KO, IKK2 KO, and IKK1/2 DKO cells. Whole-cell lysates were immunoblotted with the indicated Abs. A dagger denotes the detection of IKK2 by potential cross-reactivity of the anti-IKK1 Ab. ( B – D ) Relative levels of expression of c- fos ( B ), c- jun ( C ), and c- myc ( D ) mRNAs measured 20, 40, and 40 min, respectively, after IL-3 stimulation of Ba/F3 parental, IKK1 KO, IKK2 KO, and IKK1/2 DKO cells. * p < 0.05; n.s., not significant, as assessed by the Kruskal-Wallis test with the Steel-Dwass test. Each dot represents the result from independent experiments ( n = 5). Means are indicated by bars.

Article Snippet: The plasmid encoding FLAG-tagged IKK1 was purchased from Addgene (15467) [ ].

Techniques: Expressing, CRISPR

Journal: Cells

Article Title: IL-3-Induced Immediate Expression of c- fos and c- jun Is Modulated by the IKK2-JNK Axis

doi: 10.3390/cells11091451

Figure Lengend Snippet: IL-3-induced IKK activation was not associated with the degradation of IκB-α followed by nuclear translocation of p65. ( A ) Phosphorylation of IKKs after IL-3 stimulation. Following IL-3 deprivation for 6 h, Ba/F3 cells were stimulated with IL-3 for the indicated times, and whole-cell lysates were immunoblotted using the indicated Abs. Long and short exposures to detect phosphorylated IKK1 and IKK2 are shown. An arrowhead indicates the band corresponding to phosphorylated IKK1 at 5 min after IL-3 stimulation. A dagger denotes the detection of IKK2 by potential cross-reactivity of the anti-IKK1 Ab. ( B ) Phosphorylation of IKKs in IKK KO cells after IL-3 stimulation. Following IL-3 deprivation for 6 h, Ba/F3 parental (WT), IKK1 KO, IKK2 KO, and IKK1/2 DKO cells were stimulated with IL-3 for 5 min. Immunoblot analysis was performed with whole-cell lysates using the indicated Abs. Arrowheads indicate the bands corresponding to phosphorylated IKK1. ( C ) Degradation of IκB-α by TNF-α but not by IL-3 stimulation. Following IL-3 deprivation, Ba/F3 cells were stimulated with IL-3 or TNF-α for the indicated durations, and whole-cell lysates were immunoblotted using the indicated Abs. ( D ) Induction of nuclear translocation of p65 by TNF-α but not by IL-3 stimulation. Following IL-3 deprivation for 6 h, Ba/F3 cells were stimulated with IL-3 or TNF-α for the indicated times and immunoblotted with Abs to the cytosol marker α-tubulin and the nuclear marker fibrillarin. Long and short exposures to detect p65 are shown.

Article Snippet: The plasmid encoding FLAG-tagged IKK1 was purchased from Addgene (15467) [ ].

Techniques: Activation Assay, Translocation Assay, Western Blot, Marker

Journal: Cells

Article Title: IL-3-Induced Immediate Expression of c- fos and c- jun Is Modulated by the IKK2-JNK Axis

doi: 10.3390/cells11091451

Figure Lengend Snippet: IKK2-mediated activation of JNK regulates c- fos and c- jun expression. ( A ) Phosphorylation of JNK after IL-3 stimulation. Following IL-3 deprivation for 6 h, Ba/F3 cells were stimulated with IL-3 for the indicated durations, and whole-cell lysates were subjected to immunoblot analysis using the indicated Abs. ( B – D ) Phosphorylation of JNK in IKK KO cells after IL-3 stimulation. Ba/F3 parental (WT), IKK1 KO, IKK2 KO, and IKK1/2 DKO cells were stimulated with IL-3 after IL-3 deprivation for 6 h. Immunoblot analysis was performed with whole-cell lysates using the indicated Abs. ( E ) Expression levels of c- fos , c- jun , and c- myc mRNAs 20 and 40 min after IL-3 stimulation in the presence of JNK-IN-8 or DMSO. Ba/F3 cells were pre-incubated with 0.5 µM JNK-IN-8 or DMSO for 6 h and then stimulated with IL-3. * p < 0.05; ** p < 0.01; n.s., not significant, as assessed by Mann-Whitney U -tests, for differences between JNK-IN-8-treated and control cells at each time point. Each dot represents the result from independent experiments ( n = 5). Means are indicated by bars.

Article Snippet: The plasmid encoding FLAG-tagged IKK1 was purchased from Addgene (15467) [ ].

Techniques: Activation Assay, Expressing, Western Blot, Incubation, MANN-WHITNEY

Journal: Chembiochem

Article Title: INH14, a Small‐Molecule Urea Derivative, Inhibits the IKKα/β‐Dependent TLR Inflammatory Response

doi: 10.1002/cbic.201800647

Figure Lengend Snippet: A) HEK293 cells transfected with an NF‐kB reporter plasmid and plasmids encoding the adaptor proteins Mal (5 ng per well) or MyD88 (5 ng per well) were incubated with INH14 (1, 10, 25 μ m ). Then, the luciferase activity was recorded, and the luciferase values were normalized to the Renilla values. B) HEK293 cells were treated as in A), but transfected with plasmids encoding IRAK1 (20 ng per well), TRAF6 (80 ng per well), TAK1/TAB1 (60 ng per well each), IKKα (100 ng per well), or IKKβ (100 ng per well). Then they were incubated with INH14 (15 μ m ), and the luciferase activity was measured as indicated in A). RU: relative units. The bars represent mean and SEM of three independent experiments. (*** p <0.001; ** p <0.01; * p <0.05 by unpaired t‐test). (−) incubation with the vehicle used to dissolve INH14 (DMSO). (–/–): Transfection with mock plasmid and incubation with the vehicle used to dissolve INH14 (DMSO).

Article Snippet: Stock solutions of the compounds were prepared in sterile DMSO (Sigma–Aldrich) at a concentration of 10 m m . Plasmids : YFP‐MyD88, Flag‐IRAK1, Flag‐IKKα, Flag‐IKKβ, and Flag‐TRIF were purchased from Addgene.

Techniques: Transfection, Plasmid Preparation, Incubation, Luciferase, Activity Assay

Journal: Chembiochem

Article Title: INH14, a Small‐Molecule Urea Derivative, Inhibits the IKKα/β‐Dependent TLR Inflammatory Response

doi: 10.1002/cbic.201800647

Figure Lengend Snippet: A) IKKα (15 ng per reaction) or B) IKKβ (20 ng per reaction) were incubated with ATP (50 or 25 μ m , respectively) and substrate peptide (0.2 ng mL −1 ) in the presence of vehicle or increasing concentrations of INH14 at room temperature for 1 h. The graphs represent the mean and SEM of two independent experiments in duplicates. The IC 50 value was obtained by fitting of the sigmoidal dose–response curve. C) Mice were intraperitoneally pretreated with INH14 (5 μg g −1 ) or vehicle (DMSO/NaCl) for 1 h. Then they were i.p. injected with P2 (2.5 μg g −1 ). Vein blood was taken at time 0 and 2 h after P2 injection. The TNFα level was quantified by means of ELISA. The statistical significance was assessed with the unpaired Student t‐test. * p <0.05.

Article Snippet: Stock solutions of the compounds were prepared in sterile DMSO (Sigma–Aldrich) at a concentration of 10 m m . Plasmids : YFP‐MyD88, Flag‐IRAK1, Flag‐IKKα, Flag‐IKKβ, and Flag‐TRIF were purchased from Addgene.

Techniques: Incubation, Injection, Enzyme-linked Immunosorbent Assay