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Fig. 2. Condensin I and II associate with chromosomes independently of each other. (A) Depletion of condensin subunits by RNAi in HeLa cells. Logarithmically proliferating cells were transfected with CAP-D2, CAP-D3, Smc2 siRNAs or with control reagents, and total cell extracts were analyzed by immunoblotting at the indicated times after the transfection. Because the targeted proteins were depleted between 48-72 hours after transfection, subsequent analyses were carried out in this time window. (B) HeLa cells were transfected with control, CAP-D2, CAP-D3 or Smc2 siRNAs for 48 hours. Following treatment with 50 ng/ml nocodazole for 12 hours, mitotic cells were collected and chromosome enriched fractions were prepared and analyzed by immunoblotting as indicated. Three serial dilutions were loaded for each sample. Ponceau S staining of core histones is shown as a loading control in the bottom panel. IB, immunoblot. (C) IF microscopy of condensin I and condensin II in CAP-D2-or CAP-D3-depleted cells. Fourty-eight hours after the transfection of indicated siRNAs, cells were fixed and stained with antibodies against CAP-D2 or CAP-D3 (gray in the first column, green in the second), and DNA was counterstained with DAPI (blue). Representative prometaphase cells are shown. Arrows indicate chromosome axes where CAP-D2 and CAP-D3 are enriched.
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