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