Mounted cells were visualized under fluorescent microscope (Leica, http://www.leica.com).(B) Subcellular localization of endogenous and exogenous UXT. 293T cells were transfected with (top) or without (bottom) FLAG-UXT. Immunofluorescentmicroscopy was performed with the indicated primary antibodies.
Alternatively, we performed immunofluorescence analysis to support this speculation. Interestingly, when transfecting cells with siRNA against UXT and stimulating them with TNF-α, there were an apparently decreased percentage of cells that displayed focused nuclear p65. Approximately 90% of control cells displayed p65 inside the nucleus upon stimulation, whereas only 35–50% displayed p65 in the case of the UXT knockdown specimen (Fig. 5 E).Consistent with these data, S329/T331-phosphorylated MDC1 was detected by immunofluorescence in both control and γ-irradiated cells (Fig 2B; note the absence of staining in cells treated with MDC1 small interfering RNA (siRNA)), confirming that SDTD-phosphorylated MDC1 is present in the absence of damage and also forms IRIF.(B) Fluorescence microscopy demonstrating a shift in the subcellular localization of GFP-Sed5 from Golgi in control (WT) strains to a partially cytosolic localization in a Δget3 strain, and both cytosolic and few large puncta in Δget1/2 strains. The GFP-Sed5 puncta in get mutants do not colocalize with the Golgi marker Anp1-RFP.
(C) Fluorescence microscopy demonstrating colocalization of GFP-Sed5 and Get3-tdRFP in cytosolic aggregates that form in a Δget1/2 background.
(B) Fluorescence microscopy of control (WT) and Δget1/2 strains expressing a broad variety of TA proteins. GFP-Scs2, GFP-Sbh1, and GFP-Ysy6 under a galactose-inducible (GAL) promoter. Cherry-Sbh2 was expressed from a plasmid under the constitutive TEF2 promoter.
(C) Fluorescence microscopy of control (WT) and Δget1/2 strains expressing two mitochondrial TA proteins, Cherry-Fis1 and Cherry-Tom22, expressed from a plasmid under the constitutive TEF2 promoter.
(A) Fluorescence microscopy showing the localization of GFP-Ubc6 and mitochondrially targeted dsRED (MTS-RFP) in a control (WT) or Δget1/2 strain.
(B) Fluorescence microscopy of a time course monitoring the subcellular localizations of the peroxisomal TA protein GFP-Pex15 as well as dsRED targeted to the mitochondria (MTS-RFP) following induction of Pex15 from a galactose inducible promoter in a control (WT), get1/2, or get3 strain.To ascertain the time in infection and the subcellular compartment at which the interaction occurs, ASFV infected cells were examined by confocal laser scanning microscopy at different times post infection (at least 30 infected cells were analyzed for each time point at 0, 6, 8 and 12 hpi).
Subcellular distribution of p30 and hnRNP-K were analyzed by immunofluorescence microscopy in ASFV or mock infected cells. ASFV infection resulted in an intensification of nuclear, but not cytoplasmic, staining of hnRNP-K when compared to uninfected cells. When nuclei of infected cells were examined in detail, nuclear hnRNP-K was detected in characteristic granular structures coincident with areas completely devoid of nucleic acid staining. Interestingly, these kind of hnRNP-K accumulation was also observed in cells transiently expressing p30 (pCMV-p30), but not in mock-infected cells, suggesting the involvement of p30 on these changes (Fig. 4) during ASFV infection.
Colocalization of p30 and hnRNP-K in the nucleus of infected cells. Cells were infected with BA71V strain and analyzed at different times post infection by confocal immunofluorescence microscopy acquiring 0.1 μm optical sections from the Z-axis. A representative image of an infected cell at 8 hpi is shown (A). p30 was detected with a monoclonal anti-p30 followed by Alexa 647-conjugated goat anti-mouse antibody (red) and hnRNP-K with an anti-hnRNP-K specific serum followed by Alexa 488-conjugated goat anti-rabbit antibody (green). Discrete spots of colocalization of both proteins (orange) can be discerned in the cell nucleus.
ASFV infection induces changes in subcellular distribution of hnRNP-K. (A) Vero cells were either infected with BA71V (1 pfu/cell) or transfected with pCMV-p30 and analyzed by immunofluorescence microscopy at 8 hpi or 24 h post transfection, respectively. ASFV protein p30 was detected with a mouse anti-p30 monoclonal antibody and rhodamine red conjugated corresponding secondary antibody (red). hnRNP-K distribution in the cell nucleus was detected with a rabbit antiserum anti-hnRNP-K and Alexa Fluor 488 conjugated corresponding secondary antibody (green). Nuclei of cells were stained with Hoechst 3332. Accumulation of hnRNP-K in infected cells as spots which are coincident with the absence of nucleic acids staining can be appreciated in BA71V infected and pCMV-p30 transfected cells.