Fig. 3: Biophysical characterization, stability and cellular uptake of NEMO mimics.
From: Modulation of virus-induced NF-κB signaling by NEMO coiled coil mimics
a The potential of NEMO mimics to inhibit vFLIP-NEMO interaction was evaluated in a TR-FRET assay by monitoring the fluorescence emission of the acceptor. The data represents the mean ± SD of two independent experiments (n = 2) each performed with at least three replicates. These studies illustrated the potency of the optimized derivative CHD3NEMO. b The specific association of the FITC-derivatized CHD3NEMO with vFLIP over full length NEMO was further probed in a fluorescence polarization assay. The data represents the mean ± SD of a single experiment performed in triplicates. This experiment was repeated twice (n = 2) with similar results. c The conformation of the coiled coil mimics was investigated by circular dichroism spectroscopy in aqueous buffer. d The conformational stability of CHD3NEMO was further evaluated in a thermal denaturation study. The circular dichroism spectra were collected at regular intervals between 5–95 °C. e CHD3NEMO was found to resist serum proteases. Peptide degradation was probed over 24 h by HPLC. This data represents mean ± SD (n = 2) f Cellular uptake of FITC-labeled CHD3NEMO into live BC-1 cells. Cells were visualized by fluorescence microscopy after 1 h incubation. Effect of temperature and 10 mM sodium azide on the cellular uptake of the NEMO mimic was also explored. Hoechst stain was used to detect the nuclei. Representative images are shown from 2 independent experiments (n = 2). Scale bar = 20 μm.
