Fig. 7.

K24 and K70 contribute to Arc protein stability. a Mouse WT and acetyl-mimic mutants (K24Q, K33Q, K55Q, K70Q) Arc-Myc-FLAG were overexpressed in different Neuro2a cultures for 16 h at which point cells were treated with CHX (50 µg ml⁻¹) to block protein synthesis. Lysates were collected at the indicated time points and Arc-Myc-FLAG protein level analyzed in every sample by western blotting with an anti-FLAG antibody. Representative western blots are presented on top and quantification of data normalized to corresponding β-actin and presented as percentage of Arc-Myc-FLAG level (±SEM) at the initial time point after CHX application (t = 0). Curve represents exponential decay fit. b Estimate of protein half-life Arc for WT Arc-Myc-FLAG and each acetyl-mimic mutant is presented in table. c WT and acetyl-mutant Arc-Myc-FLAG polyubiquitination was determined in lysates from overexpressing Neuro2a cells. Lysates were immunoprecipitated with anti-FLAG and western blotted with anti-Ub or anti-Arc as indicated. The proteasome inhibitor MG132 (10 µM) was added for 20 min before preparation of lysates to allow accumulation of polyubiquitinated Arc-Myc-FLAG