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A
Lysates of nocodazole‐arrested mitotic HeLa cells transiently expressing HP1α‐Flag and Haspin‐GFP were subjected to immunoprecipitation followed by immunoblotting with the indicated antibodies.
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B
Lysates of nocodazole‐arrested mitotic HeLa cells transiently expressing SFB‐Haspin and HP1α‐GFP (WT and the indicated mutants) were subjected to immunoprecipitation followed by immunoblotting. S. exp., short exposure.
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C
Lysates of nocodazole‐arrested mitotic HeLa cells transiently expressing Haspin‐GFP and HP1α‐Flag (WT and the indicated mutants) were subjected to immunoprecipitation followed by immunoblotting.
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D
Purified MBP‐HP1α‐6xHis was subjected to pulldown by GST or GST‐Haspin‐N350, followed by anti‐HP1α immunoblotting and CBB staining. Note the presence of a significant amount of partial GST‐Haspin protein.
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E
Purified MBP‐HP1α‐6xHis was subjected to pulldown by GST or Haspin (80‐130)‐GST for 1 h, followed by anti‐MBP immunoblotting and CBB staining.
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F
Lysates of nocodazole‐arrested mitotic HeLa cells transiently expressing HP1α‐Flag and Haspin‐GFP (WT or ΔPxVxL) were subjected to immunoprecipitation followed by immunoblotting. L. exp., long exposure.
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G
Lysates of asynchronous HeLa, Haspin KO clone D2 and HP1 DKO clone 2A4 stably expressing the indicated fusion proteins were immunoblotted.
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H, I
The indicated stable cell lines were exposed to MG132, then fixed at the indicated time points for DNA staining, and quantified in around 200 cells. Means and ranges are shown (H; n = 2). After treatment with MG132 for 6 h, using mitotic chromosome spreads, the percentage of cells with cohesion loss was determined in around 100 cells (I).
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J
Lysates of asynchronous HeLa, Haspin KO clone D2 cells with or without stable expression of Haspin‐GFP (WT or ΔPxVxL) were immunoblotted.
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K
The indicated stable cell lines were analyzed for metaphase chromosome alignment in around 200 cells as in (H). Means and ranges are shown (n = 2).
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L
The indicated stable cell lines were analyzed for sister‐chromatid cohesion in around 100 cells as in (I).
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