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. 2012 Jan 1;23(1):59–70. doi: 10.1091/mbc.E11-06-0487

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© 2012 Chu et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 2: — α-pS661 detects phospho-UHRF1 in vivo and phosphorylation is eliminated when Ser-661 is changed to glycine. (A) HCT116 cells transfected with empty plasmid (CONTROL), FLAG epitope–tagged UHRF1 (UHRF1), or UHRF1 in which Ser-661 is changed to an alanine (UHRF1^S661A). α-pS661 specifically recognizes a protein of appropriate size in FLAG-UHRF1 cells, but not in FLAG-UHRF1^S661A cells, whereas both FLAG-UHRF1 and FLAG-UHRF1^S661A are recognized by α-UHRF1 and α-FLAG. (B) Endogenous UHRF1 is phosphorylated on Ser-661. HCT116 cell lysates blotted with Preimmune (PI) or α-pS661 sera that was preincubated with either the phosphorylated or a cognate nonphosphorylated form of the immunizing peptide. Actin used as a loading control. (C) Lysates from HCT116 cells were immunoprecipitated with anti-UHRF1 antibody and then treated with or without λ-phosphatase. Immunoblotting (IB) with α-UHRF1 and with α-pS661 shows that phosphatase treatment reduces recognition of UHRF1 by α-pS661 but not by α-UHRF1.