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. 2015 Jan 15;29(2):157–170. doi: 10.1101/gad.251785.114

Figure 2.

Figure 2.

PTEN is ADP-ribosylated by tankyrases in vitro and in vivo. (A,B) PTEN is PARylated in vivo. (A) HCT116 cells were lysed with NETN buffer containing PARG inhibitor ADP-HPD (5 μM) and protease inhibitor. Lysates were then immunoprecipitated using control or anti-PAR antibodies and immunoblotted using the indicated antibodies. (B) HCT116 cells were lysed with NETN denaturing buffer containing PARG inhibitor ADP-HPD (5 μM) and protease inhibitor. Lysates were then immunoprecipitated using control or anti-PTEN antibodies followed by Western blotting as indicated. (C) Ribosylation of PTEN by TNKS1 and TNKS2 in vitro. Recombinant TNKS1, TNKS2, and PTEN were subjected to in vitro ribosylation assays in the absence or presence of biotin-labeled NAD+. The recombinant proteins were detected by the indicated antibodies, and the ribosylated proteins were determined with anti-biotin antibody. (D) The ribosylation of PTEN by TNKS1 is diminished by tankyrase inhibitor XAV939. The recombinant MBP-PTEN and TNKS1 were subjected to an in vitro ribosylation reaction as described above in the absence or presence of the indicated concentrations of XAV939. (E) The catalytic activity of TNKS1 is required for PTEN ribosylation. The MBP-PTEN and immunoprecipitated SFB-tagged wild-type or the catalytically inactive mutant of TNKS1 (TNKS1-PD) were subjected to an in vitro ribosylation reaction followed by Western blotting as indicated. (F) The tankyrase-binding motif of PTEN is required for its ribosylation by TNKS1. The recombinant MBP-PTEN, MBP-PTEN-AA, and TNKS1 were subjected to an in vitro ribosylation assay and analyzed by Western blotting as indicated. (G) Only double knockdown of TNKS1/2 diminishes the ribosylation of PTEN in vivo. HCT116-derived cells with stable knockdown of TNKS1, TNKS2, TNKS1/2, or PTEN were collected and immunoprecipitated using anti-PTEN antibody. The input and immunoprecipitated proteins were analyzed by Western blotting using the indicated antibodies, and the ribosylation of endogenous PTEN was detected by anti-PAR antibody.