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. 2016 Jun 30;7(31):50215–50228. doi: 10.18632/oncotarget.10330

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Copyright: © 2016 Kreis et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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A. Pulldown assays were performed with nocodazole treated cellular extracts from HCT116 p21−/− cells in the presence of GST, GST-p21, GST-p21S130A or GST-p21S130D. B. HCT116 p21−/− cells were transfected with wild type FLAG-p21, FLAG-p21S130A or FLAG-p21S130D and synchronized to prometaphase with nocodazole. The amounts of transfected plasmids were adjusted, so that the expression levels of FLAG-p21 and its mutants were comparable. Left panel: whole cell extracts and nuclear extracts were prepared for Western blot analysis as transfection efficiency and input control for immunoprecipitation in the right panel. Calnexin served as cytoplasmic control. Right panel: immunoprecipitation of FLAG-transfected nuclear cell extracts with cyclin B1 antibody. Light chain served as loading control. C. HeLa cells transfected with wild type FLAG-p21 or its mutants were non-treated (con) or synchronized to prometaphase with nocodazole (noc) and cytoplasmic (cy) and nuclear extracts (nu) were prepared for Western blot analysis. Calnexin and lamin B1 served as cytoplasmic and nuclear extract marker, respectively.