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. 2008 May 28;3(5):e2277. doi: 10.1371/journal.pone.0002277

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McNally et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) Immunolocalization of PML Ib. PML ^+/+ PML ^−/−, and B1041-1 mouse fibroblast cell lines transfected with a human PML Ib/pcDNA3.1 construct were immunostained with PG-M3 to determine the intracellular localization of PML Ib by confocal (PML^+/+) and indirect (PML^−/− and B1041-1) immunofluorescence microscopy. DAPI was used to counter-stain the nucleus. (B) Localization of PML isoforms I, IV, and VI was revealed by immunostain with PG-M3 and Texas Red following the transfection of each cDNA into B1041-1 cells. (C) Co-transfection of PMLIb with nuclear PML isoforms induces a redistribution of PML protein to the cytoplasm: PMLIb+PML I (stable transfectant, left image); PMLIb+PML IV (transient transfection, middle image); PMLIb+PML VI (transient transfection, right image). Cells were immunostained with PG-M3 and Texas red conjugated secondary antibody as above. Note that the cells transiently co-transfected with PML Ib+PML VI (leftmost image) are dual stained with PG-M3 and a murine polyclonal PML antibody and show re-localization of endogenous mouse PML. DAPI was used to counter-stain the nucleus. Localization of PML was examined by indirect immunofluorescence microscopy. (D & E) Interaction between nuclear and cytoplasmic PML isoforms. V5-tagged PML I/ pcDNA 4, myc-tagged PML Ib/pcDNA 3.1 and vector constructs were transiently transfected either alone with vector or in combination, into the HEK293 cell line. 48h post-transfection cells were (D) lysed and subjected to co-immunoprecipitation or (E) fixed and immunostained. (D) V5 and myc antibodies were used for immunopreciptiations while HRP-conjugated V5 and myc antibodies were used for immunoblots. As shown in the bottom right panel, myc-tagged PML Ib co-precipitated with V5-tagged PMLI, whereas transfection of PML I or PML Ib alone did not result in co-immunoprecipitation. Panels on the left (top & bottom) represent immunoblots of the crude lysates (10 µg protein/lane), and are included to show the antibody specificity for the V5-tagged PML I and Myc-tagged PML Ib constructs. V = vector control, I = PML I, Ib = PML Ib, I+Ib = co-transfected PML I & PML Ib. (E) Cells co-transfected with both vectors, PML I-V5+pcDNA 3.1-myc, PML Ib-myc+pcDNA 4-V5-his, or PML I-V5+PML Ib-myc and grown on glass cover slips were single stained with a FITC conjugated V5 antibody (bottom two panels), or the anti-myc antibody, 9E10, and anti mouse Texas Red (top two panels) as indicated in the figure.