Figure 1.
Subcellular localization of GFP fused with the short or long N-terminal sequence of Ilf3/NF90.
Plasmids pEGFP-N1 (Control, left panels), pEGFP-N1-Ilf3/NF90 common N-terminal short sequence (Short-GFP, mid panels) and pEGFP-N1-Ilf3/NF90 common N-terminal long sequence (Long-GFP, right panels) were transfected into HeLa cells. After 24 hours, cells were fixed and co-stained with anti-α-tubulin antibody (α-Tub) and DAPI. GFP or GFP fusion proteins appear in green, α-tubulin in red and DAPI staining in blue. Arrows point to intranuclear foci.
Figure 2.
Subcellular distribution of Ilf3 and NF90 in P19 cells.
After subcellular fractionation, proteins from identical percentages of each fraction were submitted to SDS-PAGE, blotted onto nitrocellulose and immunodetected with the serum Ab78 raised against Ilf3 and NF90 (S.E.: short exposure time; L.E.: long exposure time), anti-UBF serum (UBF) or anti-α-tubulin antibody (α-tub.). Molecular weight markers (kDa) are indicated at the right.
Figure 3.
Ilf3 and NF90 polymorphism in nuclear fractions purified from P19 cells.
Ilf3 and NF90 from P19 cell nuclear fractions were submitted to 2-D PAGE and immunodetected with polyclonal antibody 78. Arrows positioned in the same coordinates in Ilf3 or NF90 panels indicate the positions of Ilf3 and NF90 long and short isoforms. The faint signal in the middle right panel was detected with a ten fold longer time exposure than that of the other panels.
Figure 4.
Subcellular localization of human eRF1 fused with short or long N-terminal sequence of Ilf3/NF90.
Plasmids pCMV-heRF1-Ilf3/NF90 short N-terminal sequence (N-heRF1, mid panels) and pCMV-heRF1-Ilf3/NF90 long N-terminal sequence (NoLS-heRF1, lower panels) were transfected into HeLa cells. After 24 hours, untransfected (Control, upper panels) or transfected cells were co-stained with anti-heRF1 antibody (heRF1), anti-α-tubulin antibody (α-Tub) and DAPI. Endogenous heRF1 or heRF1 recombinant fusion proteins appear in green, α-tubulin in red and DAPI in blue. Arrows point to intranuclear foci corresponding to nucleoli.
Figure 5.
Subnuclear distribution of GFP fused to deletion or substitution mutants of Ilf3/NF90 NoLS.
(A) Plasmids pEGFP-N1 (Control), pEGFP-N1-Ilf3/NF90 common N-terminal long region (NoLS-GFP), pEGFP-N1-Ilf3/NF90 common N-terminal short region (N-GFP), pEGP-N1/deletion mutant of the four-arginine stretch from the NoLS (Δ4R-GFP), pEGP-N1/substitution mutant of the threonine 10 by alanine (T10A-GFP) and pEGP-N1/substitution mutant of the threonine 10 by aspartate (T10D-GFP) and (B) plasmids pEGP-N1/deletion mutant of the three-histidine stretch (Δ3H-GFP), pEGP-N1/substitution mutant of the three-histidine stretch by three alanines (3H->3A-GFP), three phenylalanines (3H->3F-GFP), three lysines (3H->3K-GFP) or three glutamates (3H->3E-GFP) and pEGP-N1/substitution mutant of the three-histidine stretch by three lysines/deletion mutant of the four-arginine stretch (3H->3K/Δ4R-GFP) were transfected into HeLa cells. After 24 hours, cells were co-stained with the monoclonal anti-B23 antibody (B23) and DAPI. After confocal microscopy acquisition, focal planes exhibiting a B23 optimal signal were chosen. GFP fusion proteins appear in green, B23 in red and DAPI in blue.
Figure 6.
Subnuclear distribution of exogenously-expressed S-Ilf3, L-Ilf3 and L-NF90 isoforms in HeLa cells.
Plasmids pEGFP-N1 (Control row), pEGFP-N1-S-Ilf3 (S-Ilf3 row), pEGFP-N1-L-Ilf3 (L-Ilf3 row) or pEGFP-N1-L-NF90 (L-NF90 row) were transfected into HeLa cells. After 24 hours, cells were co-stained with human anti-fibrillarin serum (Fibrillarin) and monoclonal anti-B23 antibody (B23). After confocal microscopy acquisition, focal planes were chosen to obtain optimal fibrillarin and B23 signals. DAPI (not shown here) was used to define the nuclear limits (white drawings). GFP and GFP fusion proteins appear in green, fibrillarin in red and B23 in blue.
Figure 7.
Subcellular distribution of exogenously-expressed L-NF90 isoforms in HeLa cells treated with DRB.
Plasmids pEGFP-N1-L-NF90 (L-NF90-GFP) and mcherry-B23 (B23-mcherry) were transfected into HeLa cells. 24 hours later, cells were fixed either immediately (Control, upper panels) or after a DRB treatment during two hours (DRB 2 h, mid panels) followed by a chase of one hour (Chase 1 h, lower panels). GFP and mcherry fusion proteins appear in green and red, respectively.
Figure 8.
Fluorescence recovery in HeLa cell nucleoli after photobleaching (FRAP) of GFP-tagged L-Ilf3, L-NF90 or B23.
Plasmids pEGFP-N1-L-Ilf3, pEGFP-N1-L-NF90 or pEGFP-N1-B23 were transfected into HeLa cells. After 24 hours, living cells were subjected to photobleaching. A. In HeLa cells expressing L-NF90-GFP, one nucleolus was targeted for laser bleaching (left panel, green circle in the upper cell) whereas another nucleolus from the same cell (blue circle in the upper cell) and a nucleolus from a distinct cell (yellow circle in the lower cell) were marked to serve as controls. Images were acquired every two seconds during 40 seconds before, immediately after or during 240 seconds after bleaching (prebleach, bleach, post-bleach 6 and 240 seconds, respectively). B. Fluorescence recordings emitted from the 3 delimited regions in A (green: bleached nucleolus; purple: control nucleolus from the same cell; yellow: control nucleolus from another unbleached cell). C. Kinetics of mean fluorescence recovery after photobleaching of GFP-tagged L-Ilf3 (n = 14), L-NF90 (n = 15) or B23 (n = 11) after a normalization of fluorescence intensity. The t1/2 of mean fluorescence recovery are indicated in the figure.
Table 1.
Occurrence frequencies (%) of possible NoLS sequences (K/R-K/R-X-K/R) and of the twenty aminoacids at the X position in several NoLS-containing nucleolar proteins.