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. 2009 Apr 8;4(4):e5118. doi: 10.1371/journal.pone.0005118

Figure 4. HP1 knock down cells show no defect in cohesin recruitment.

Figure 4

(A) HeLa cells transfected with no siRNA (control, lanes 1–4) or siRNAs specific to HP1alpha (lanes 5–8), HP1beta (lanes 9–12) or HP1gamma (lanes 13–16), were separated in three fractions: a soluble cytoplasmic fraction (Sc), a soluble nucleoplasmic fraction (Sn) and a chromatin-enriched fraction (Chr). T is total cell extract. ORC2 and MEK2 are chromatin-bound and cytoplasmic proteins, respectively, that serve as control for the fractionation protocol. (B) A HeLa cell line expressing Scc1-9xmyc under the control of doxicycline was transfected with siRNAs against HP1alpha, HP1gamma, or both, and the remaining levels of each protein were assayed by quantitative immunoblotting five days after transfection. (C) The same cells were grown on coverslips, pre-extracted before fixation and stained with anti-myc (red) and CREST serum (green) and counterstained with DAPI (blue). As expected, the Scc1-myc signal appears between the two sisters centromeres labeled by CREST (inset). Scale bars, 5 micrometers and 1 micrometer (inset on the left). (D) Fraction of mitotic cells showing the staining depicted in (C) relative to control cells (see materials and methods).