Journal: bioRxiv

Article Title: BRD4 regulates Aurora B kinase activity

doi: 10.1101/2025.08.06.668960

Figure Lengend Snippet: A. Aurora B interaction with BRD4 is unaffected by its phosphorylation. Co-IP of Aurora B with Flag-BRD4 on Flag beads following a kinase assay with or without ATP. B. JNK activation and phosphorylation of BRD4 in vivo abrogates BRD4-Aurora B interaction. Immunoblots showing Aurora B co-immunoprecipitated by anti-BRD4 with total and T1212 phosphorylated BRD4 from HCT116 cells treated with or without JNK activator anisomycin. C. JNK activation increases MCAK Ser95 phosphorylation. Immunoblots showing MCAK pSer95 and pJNK levels in HCT 116 cells with or without JNK activation by anisomycin for increasing durations of time. Data is representative of two independent experiments. D. JNK inhibition decreases MCAK Ser95 phosphorylation. Immunoblots showing MCAK pSer95 levels in HCT116 cells with or without anisomycin and JNK inhibitor D-JNK1. E. JNK does not directly phosphorylate MCAK or affect Aurora B activity. Autoradiograph of kinase assays with equimolar Aurora B and active JNK with MCAK as substrate. F. MCAK phosphorylation is dependent on JNK phosphorylation of BRD4. Immunoblots showing MCAK pSer95 levels in DLD-BRD4-IAA7 cells transfected with or without WT or 3A-BRD4 and treated with or without auxin and anisomycin.

Article Snippet: Purified recombinant kinase active human GST-tagged Aurora B and JNK1 was purchased from SignalChem.

Techniques: Phospho-proteomics, Co-Immunoprecipitation Assay, Kinase Assay, Activation Assay, In Vivo, Western Blot, Immunoprecipitation, Inhibition, Activity Assay, Autoradiography, Transfection

Journal: The Journal of biological chemistry

Article Title: Interleukin-1 beta-induced ceramide and diacylglycerol generation may lead to activation of the c-Jun NH2-terminal kinase and the transcription factor ATF2 in the insulin-producing cell line RINm5F.

doi: 10.1074/jbc.271.14.8307

Figure Lengend Snippet: FIG. 3. A, effects of acetylsphingosine, PMA, and IL-1b on JNK1 phosphorylation. RINm5F cells were 32P-labeled for 2 h and then ex- posed to 10 mM acetylsphingosine (lane 2), 100 nM PMA (lane 3), and 25 units/ml IL-1b (lane 4) for 20 min. JNK1 was immunoprecipitated and analyzed by SDS-polyacrylamide gel electrophoresis. The position of JNK1 (46 kDa) is indicated by the lower arrow. The upper arrow indicates the position of the 54-kDa JNK2 phosphoprotein. This figure is representative of three separate experiments. B, effects of acetyl- sphingosine, PMA, and IL-1b on JNK1 in vitro kinase activity. RINm5F cells were exposed for 20 min to 10 mM acetylsphingosine (lane 2), 100 nM PMA (lane 3), 10 mM acetylsphingosine 1 10 nM PMA (lane 4), and 25 units/ml IL-1b (lane 5). Lane 1 in A and B is the control. Homoge- nates were then immunoprecipitated for JNK1, and the in vitro kinase activity was determined using GST-c-Jun fusion protein as substrate. The position of phosphorylated GST-c-Jun is indicated by the arrow. C, Coomassie Brilliant Blue staining of cell homogenates separated by SDS-polyacrylamide gel electrophoresis. Lanes are the same as de- scribed for B.

Article Snippet: Effects of IL-1b, Acetylsphingosine, and PMA on JNK1 Phos- phorylation and JNK1 Kinase Activity—The Santa Cruz JNK1 (N-19) antibody is reactive mainly against the 46-kDa JNK1 protein, but cross-reacts also with the 54-kDa JNK2 protein.

Techniques: Phospho-proteomics, Labeling, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, In Vitro, Activity Assay, Control, Staining