Quantitative BiFC analysis of Nipah, Hendra, Sendai and Mumps virus matrix ubiquitination regulated by a lysine within the NLS_bp.

<p><b>(A-D)</b> Bimolecular fluorescence complementation (BiFC) analysis of ubiquitination of WT or NLS_bp-lysine mutants of <b>(A)</b> NiV-M, <b>(B)</b> HeV-M, <b>(C)</b> SeV-M and <b>(D)</b> MuV-M. Top, Extended Focus (maximum intensity projection) view of 3D confocal micrographs of HeLa cells cotransfected with VN173-Ub and WT, or the indicated NLS_bp-lysine mutants of VC155-tagged NiV-M, HeV-M, SeV-M or MuV-M. At 24h post-transfection, cells were counterstained with DAPI to visualize nuclear DNA, blue, and fluorescent phalloidin to visualize the F-actin cytoskeleton, red. BiFC fluorescence is pseudocolored green. Scale bar 10 μm. Middle, the background subtracted BiFC fluorescence per cell was quantified from 3D-reconstructed confocal micrographs. *p<0.05; ***p<0.001; ****p<0.0001 by a Mann-Whitney test. <b>(E-H)</b> Control immunoblots comparing expression levels of WT or NLS_bp-lysine mutant of VC155-tagged <b>(E)</b> NiV-M, <b>(F)</b> HeV-M, <b>(G)</b> SeV-M and <b>(H)</b> MuV-M in whole cell lysates (WCL) of transfected HeLa cells.