Journal: Nature Communications
Article Title: Cross-regulation of viral kinases with cyclin A secures shutoff of host DNA synthesis
doi: 10.1038/s41467-020-18542-1
Figure Lengend Snippet: a Sequence alignment of overlapping NLS-RXL elements in M97 and U69 kinases. Stretches of basic residues characteristic for nuclear localization signals (NLS) are highlighted in blue, conserved leucine and phenylalanine residues participating only in the RXL/Cy motif in pink. A mutation strategy was designed to separate NLS function from cyclin binding. RXL-AXA mutations affect both RXL and NLS sequences, LXF-AXA and LF-AA mutations only the RXL/Cy motif. b HEK-293T cells were transfected with HA-tagged wild-type (WT) or mutant forms of U69 and M97 kinases. Cyclin A co-IP was carried out at 2 days post transfection and analyzed by immunoblotting for the presence of viral kinases and Cyclin A. The immunoblots are representative of three independent experiments with similar results. c Sequence fragments encompassing the NLS-RXL region of U69/M97 mutant and wild-type kinases were cloned between and in-frame with the green fluorescent protein (GFP) and β-galactosidase genes in pHM830. d , e 293T cells were transfected with the M97/U69-NLS reporter constructs, as indicated. The subcellular localization of GFP was analyzed at 24 h post transfection using confocal live-cell imaging microscopy. Nuclei were counterstained with Hoechst-33342. Scale bars: 10 μm. e The indicated number of cells were categorized based on the subcellular localization of the GFP reporter relative to the Hoechst stain.
Article Snippet: PHM830 (Addgene plasmid #20702) was a gift from Thomas Stamminger .
Techniques: Sequencing, Mutagenesis, Binding Assay, Transfection, Co-Immunoprecipitation Assay, Western Blot, Clone Assay, Construct, Live Cell Imaging, Microscopy, Staining
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