Fig 2: IL-3-induced IKK activation was not associated with the degradation of I?B-a 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 I?B-a by TNF-a but not by IL-3 stimulation. Following IL-3 deprivation, Ba/F3 cells were stimulated with IL-3 or TNF-a for the indicated durations, and whole-cell lysates were immunoblotted using the indicated Abs. (D) Induction of nuclear translocation of p65 by TNF-a but not by IL-3 stimulation. Following IL-3 deprivation for 6 h, Ba/F3 cells were stimulated with IL-3 or TNF-a for the indicated times and immunoblotted with Abs to the cytosol marker a-tubulin and the nuclear marker fibrillarin. Long and short exposures to detect p65 are shown.

Fig 3: 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.

Fig 4: 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.

Fig 5: A putative model for IKK-mediated IL-3 signaling to induce expression of IEGs.