Journal: Cells
Article Title: Ubiquitin Activating Enzyme UBA6 Regulates Th1 and Tc1 Cell Differentiation
doi: 10.3390/cells11010105
Figure Lengend Snippet: UBA6 is required for the inhibition of NF-κB activation in CD8 + T cells. Sorted naïve CD8 + T cells were activated with soluble anti-CD3 and anti-CD28 Ab for the indicated times. ( A , B ) Cells were activated in the presence of 50 μM cycloheximide. Whole cell lysates were prepared and analyzed by western blot analysis using p-IκBα, IκBα, and UBA6 Abs to detect these proteins after ( A ) shot time and ( B ) long time stimulation with anti-CD3 and anti-CD28 Ab. ( C ) Flow cytometry analyses of phospho-p65 the levels in CD4 and CD8 T cells. Cells were unstimulated (left) or stimulated for 15 min (right) (left panel). MFI of phosphor-p65 levels are shown (right panel) ( n = 4, two-way ANOVA, mean ± SEM, ** p < 0.01). ( D ) Nuclear transcription factor NF-κB p65 activity was measured by ELISA-based TransAM NF-κB p65 kit (Active Motif) according to the manufacturer’s protocol. Data are representative of three independent experiments.
Article Snippet: To detect transcription factors (phospho-p65 and phospho-c-Jun), we followed the manufacturer’s protocol from eBioscience (San Diego, CA, USA).
Techniques: Inhibition, Activation Assay, Western Blot, Flow Cytometry, Activity Assay, Enzyme-linked Immunosorbent Assay