Figure 4
From: GSK3β N-terminus binding to p53 promotes its acetylation
GSK3 regulates p53 acetylation which regulates association with GSK3β. (A) SH-SY5Y cells were preincubated with 1 μM TSA and 5 mM nicotinamide (T/N) for 2 hr and treated with 1 μM camptothecin (CA) for 3 hr. Total p53 was immunoprecipitated followed by immunoblotting with antibodies specific for acetylated Lys373 (Ac-K373-p53), Lys382 (Ac-K382-p53), Lys320 (Ac-K320-p53) (Trevigen), total p53 (Santa Cruz), and β-actin (Sigma). (B) Quantitative analysis of the percentage of acetylated p53. SH-SY5Y cells were preincubated with 1 μM TSA and 5 mM nicotinamide for 2 hr, with GSK3 inhibitors, 20 mM lithium (Li), 10 μM SB216763 (SB), or 10 μM CHIR99021 (CHIR), for 30 min, and treated with 1 μM camptothecin for 3 hr. Quantitative values are means ± S.E.; n = 3. *p < 0.05, ANOVA with Dunnett's post hoc multiple comparisons test. (C) SH-SY5Y cells were preincubated with 1 μM TSA and 5 mM nicotinamide (T/N) for 2 hr and treated with 1 μM camptothecin (CA) for 3 hr. The association of GSK3α/β with p53 was assessed by immunoprecipitation of GSK3α or GSK3β followed by immunoblotting total p53. The endogenous total p53, total GSK3α/β, acetylated p53 (Ac-K373-p53, Ac-K382-p53, Ac-K320-p53), and β-actin levels were determined by immunoblotting.