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. 2004 Mar 12;101(12):4030–4035. doi: 10.1073/pnas.0306848101

Fig. 4.

Fig. 4.

HPV-11 GFP-E2K239A colocalizes with MTOC and microtubules at interphase and with centrosomes and mitotic spindles at metaphase. (AC) COS7 cells were transfected with pGFP-H11 E2K293A. At 16 h, nuclear DNA was DAPI-stained (blue) and the fusion protein was revealed by using GFP. (A) An interphase cell with high fusion protein expression. (B) The bright dot in interphase cells was positive for γ-tubulin (Cy-3), indicative of MTOC. (C) A metaphase cell expressing a very low level of GFP fusion protein. The spindle poles are the only clearly GFP-positive structures. The image is enlarged relative to A and B for clarity. (DF) A metaphase cell expressing GFP-11 E2K239A (D) was probed for β-tubulin (Cy-3) (E); shown is a merged image (F) with the metaphase chromosomes (DAPI). The centrosomes and spindles were decorated by the GFP fusion, which was clearly excluded from the chromosomes. (GN) A time course [0 min (G and H), 5 min (I and J), 10 min (K and L), and 15 min (M and N)] of microtubule repolymerization at 37°C after being dissociated at 4°C. β-Tubulin was revealed by indirect immunofluorescence (Cy-3). (H, J, L, and N) Merged images. Aster formation can be seen over time from the MTOC, the bright GFP/tubulin focus. (O) The GFP-H11 E2K239A-STR protein was localized to microtubules and centrosomes in an interphase cell. Shown are GFP (Left), β-tubulin (Cy-3) (Center), and merged images with chromosomal DNA stained with DAPI (blue) (Right). (P) α-, β- and γ-tubulins were pulled down with streptavidin-conjugated Sepharose beads from COS7 cells transfected with an expression vector of GFP-H11 E2K239A-STR (lanes 3) but not from mock-transfected cells (lanes 2). Western blots were probed with individual antibodies to the tubulins (Lower) and then stripped and reprobed for E2 by using a GFP antibody (Upper). Lanes 1 show a small fraction of whole cell lysates from mock-transfected cells.