Figure 3.

AceTAG targeted acetylation can be applied to multiple POIs. (a) AceTAG-1 induces acetylation of FKBP12^F36V-p65/RelA fusion in HeLa cells. FKBP12^F36V-RelA-HA RelA−/− HeLa cells were treated with increasing concentrations of AceTAG-1 (2 h) and lysed, and FKBP12^F36V-RelA was enriched and blotted with noted antibodies. Shown in the right panel is quantitation of immunoblot signal for p65 K310Ac relative to FKBP12^F36V-RelA-HA as the mean ± s.e.m. of n = 2 biologically independent experiments. (b) Immunoblot analysis of controls for AceTAG-mediated acetylation of RelA. Cells were treated with TSA (2 μM) and nicotinamide (10 mM) as a positive control for p65 acetylation⁴² or co-treated with AceTAG-1 (625 nM) and DMSO, an FKBP12^F36V binding ligand (Figure S1c), or the p300/CBP KAT domain inhibitor A485 for 2 h which block AceTAG-1-induced acetylation. (c) AceTAG-1 induces acetylation of FKBP12^F36V-p53 fusion in H1299 cells. FKBP12^F36V-p53HA H1299 was treated with increasing concentrations of AceTAG-1 (2 h) and lysed, and FKBP12^F36V-p53 was enriched and blotted with noted antibodies. Shown in the right panel is quantitation of immunoblot signal for p53 K382Ac relative to FKBP12^F36V-p53-HA as the mean ± s.e.m. of n = 3 biologically independent experiments. (d) Immunoblot analysis of controls for AceTAG-mediated acetylation of p53. The results in (b, d) are representative of two independent biological replicates (n = 2). Full images of blots are shown in Figures S9 and S10. Statistical significance (black asterisks) was calculated with unpaired two-tailed Student’s t tests comparing DMSO- to AceTAG-1-treated samples. Statistical significance (red asterisks) of the autoinhibition effect was calculated with unpaired two-tailed Student’s t tests comparing 625 nM AceTAG-1- and 15 or 50 μM AceTAG-1-treated samples. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.