Fig 2. ML sequesters TFIIB from the nucleus, but does not cause its degradation.

A) Confocal immunofluorescence analysis of HeLa Kyoto cells stably expressing GFP-TFIIB infected with THOV wt, THOV-ΔML or THOV-SW mutants for 24 hours at MOI 3. HeLa cells were treated as indicated, fixed and stained with GFP-DyLight488, THOV NP+rbAlexa546 and DAPI and subjected to confocal microscopy. Images are representative of two independent experiments with similar results. White bar – 10 μm. B) Confocal immunofluorescence analysis of HeLa Kyoto cells stably expressing GFP-TFIIB and transiently transfected with HA-M or HA-ML for 16 hours. HeLa cells were treated as indicated, fixed and stained with GFP-DyLight488, HA+msAlexa594 and DAPI and subjected to confocal microscopy. Images are representative of four independent experiments with similar results. White bar – 10 μm. C) Cytoplasmic-nuclear fractionation of Vero cells transiently transfected with HA-tagged M or ML for 24 hours. Depicted is endogenous TFIIB. Western blot is a representative of three experiments with similar results. D) Cytoplasmic-nuclear fractionation of Vero cells transiently transfected with FLAG-TFIIB and HA-tagged M or ML for 24 hours. Western blot is a representative of three experiments with similar results. E) Total protein intensity of TFIIB as defined by iBAQ, levels of newly synthesized TFIIB as determined from heavy intensities, and translation rates of identified proteins determined from H (newly synthesized)/L (total) ratios presented as box-whisker plots with whiskers showing 10–90 percentile. Protein levels were estimated in four biological replicates.