FAM134B is acetylated at lysine160 (K160), which is responsive to ER stress. (A) Representative MS/MS spectrum of acetylated peptide 149SLSESWEVINSK(Ace)PDERPR166 (m/z = 533.27, MH₄⁴⁺), which was acetylated at lysine160 (K160) of FAM134B. (B) Specificity verification of recognizing FAM134B K160 acetylation (~70 kD). Mutation of lysine (K) to arginine (R) mimicking deacetylation, which abolished recognition by Ace-K160) antibodies. HEK293T cells were transfected with FAM134B-Flag. IP was performed with anti-Flag beads. FAM134B K160 acetylation was analyzed by Western blot (WB) with Ace-K160 antibodies. (C) K160 acetylation of endogenous FAM134B. IP was performed with an antibody to acetylated lysine. The control was the Protein A/G agarose resin without the antibody that can recognize acetylated FAM134B. K160 acetylation and FAM134B were detected by Western blot. (D and E) K160 acetylation of endogenous FAM134B in response to ER stress. HEK293T cells were treated with EBSS starvation medium or 1 μM of Tg to trigger ER stress for 0, 0.5, 1 h. IP was performed with an antibody to acetylated lysine. K160 acetylation, FAM134B, p-PERK, and PERK were detected by Western blot. (F) K160 acetylation of endogenous FAM134B increased by treatment with deacetylase inhibitors. HEK293T cells were treated with deacetylase inhibitors TSA (1 mM) and NAM (5 mM) for 12 h. The control was treated with DMSO. IP was performed with an antibody to acetylated lysine. K160 acetylation and FAM134B were detected by Western blot. Molecular weight measurements are in kD. Source data are available for this figure: SourceData F1.