Research Paper Volume 8, Issue 12 pp 3356—3374

Figure 7. Perturbation of NonO-PSF stoichiometry induces nuclear to cytoplasmic relocalization during senescence induction. Up- (indicated by ↑) or down (↓)-modulation of NonO levels leads to nuclear exit and altered stoichiometry of NonO (A) and PSF (B). Human BJ diploid fibroblasts were transfected with NonO (↑) or with NonO-AS (↓) and whole cell lysates were prepared just prior (day 8) to when senescence is observed (Fig. 1D). Nuclear and cytoplasmic extracts were prepared and then fractionated by Superose 6 FPLC under high salt conditions (400 mM KCl) to determine PSF (P) and NonO (N) elution profiles. Approximate masses in each fraction were assessed relative to the mobility of ferritin (440 kDa), catalase (232 kDa), lactate dehydrogenase (140 kDa), and albumin (66 kDa) run on parallel columns (Suppl. Fig. 6). Each fraction (indicated by numbers below the lanes) was concentrated and analyzed by Western blotting with anti-NonO (A) and PSF (B) antibodies. PSF (~100 kDa) and NonO (~50 kDa) elute either as monomers (P or N) or as part of a larger complex corresponding to homodimers (P2 or N2), heterodimers (P+N) and heterotetramers (P2+N2).