Fig. 2. p53 is crotonylated at Serine 46.

(A) Ectopic p53 is crotonylated. p53, p63 and p73β plasmids were introduced into HCT116^p53−/− cells. Cells 30h after transfection (the same time for the following IP-IB experiments) were harvested for IP-IB or straight IB analysis with indicated antibodies. (B) Endogenous p53 is crotonylated. HCT116 cells were used for IP with the anti-p53 antibody followed by IB with indicated antibodies. (C) The N-terminus of p53 is crotonylated. Deletion mutants of p53 were introduced into HCT116^p53−/− cells. Cells were harvested for IP-IB with indicated antibodies. (D) p53-K24R is crotonylated. WT p53 and p53-K24R plasmids were introduced into HCT116^p53−/− cells. Cells were harvested for IP-IB with indicated antibodies. (E) p53–4M is not crotonylated. WT p53 and p53–4M plasmids were introduced into HCT116^p53−/− cells, which were harvested IP-IB with indicated antibodies. (F) p53-S46A is not crotonylated. The S33A, S46A, T81A or 4M mutants of p53 were introduced into HCT116^p53−/− cells that were harvested for IP-IB with indicated antibodies. (G) p53 is crotonylated in vitro. His-p53WT or His-p53S46A was purified from E.coli for in vitro enzyme reactions using HEK293T cell lysate and Cro-CoA. Crotonylated proteins were detected by IB with the anti-pan cro antibody (G). (H) Crotonylation reactions were performed with WT and S46A(SA) His p53 beads in the presence and absence of HEK293 lysate. Crotonylation was detected using a TCEP-Biotin probe was used in the in vitro reaction and a high sensitivity HRP-Streptavidin antibody was used for detection of crotonylation. (I) CA does not alter murine p53 level. MEF cells were treated with different doses of CA (1,5mM) for 24h as indicated and harvested for IB with indicated antibodies. (J) CA induces crotonylation, but reduces Ser46 phosphorylation, of p53. HCT116 cells were treated with etoposide and AA or CA for 24h and harvested for IP-IB analysis with indicated antibodies.