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. 2023 May 1;133(9):e164528. doi: 10.1172/JCI164528

Figure 1. Acetylation at Lys 98 promotes FGL1 degradation via a ubiquitin-proteasome pathway.

Figure 1

(A) Immunoblot (IB) analysis of Flag-FGL1 acetylation in HEK293T cells stably expressing Flag-FGL1 in the presence or absence of the deacetylase inhibitors TSA (5 μM, 16 h) and NAM (10 mM, 6 h). FGL1 acetylation was detected with an anti–acetylated lysine antibody. (B) IB analysis of whole-cell lysates (WCLs) and anti-lysine immunoprecipitates derived from HCCLM3 cells with IgG as a negative control. (C) IB analysis of acetylated, stably expressed Flag-FGL1 in HEK293T cells treated with TSA (5 μM, 16 h), NAM (10 mM, 6 h), or both. (D) Alignment of amino acid sequences of FGL1 containing Lys 98 across species. (E) The specificity of the site-specific anti–FGL1 acetylation antibody was determined by dot blot. (F) IB analysis of FGL1 acetylation derived from HEK293T cells transfected with WT Flag-FGL1 or the K98R mutant. FGL1–Non-Ac, nonacetylated FGL1. FD, fibrinogen domain; SP, signal peptide; CCD, the coil-coil domain. (G) IB analysis of WCLs and anti-Flag IPs derived from HEK293T cells transfected with the indicated constructs. (H) IB analysis of endogenous FGL1 in multiple HCC cell lines treated or not with NAM (10 mM, 6 h). (I) qPCR analysis of relative FGL1 mRNA levels in multiple HCC cell lines treated or not with NAM. Data indicate the mean ± SD of 3 independent experiments. CON, control. (J IB analysis of endogenous FGL1 in HCCLM3 cells treated with NAM in the presence or absence of the proteasome inhibitor MG132, the lysosome inhibitor NH4Cl, or the autophagy inhibitor 3-MA. (K) IB analysis of FGL1 in HEK293T cells transfected with WT Flag-FGL1 or K98 mutant plasmids with or without NAM treatment. (L) WT Flag-FGL1 and the K98 mutants were cotransfected with HA-Ub into HEK293T cells with or without NAM treatment. The level of FGL1 ubiquitylation was determined by IB analysis. (M) IB analysis of turnover rates in HEK293T cells stably expressing WT Flag-FGL1 or the K98R or K98Q mutants treated with 100 μg/mL cycloheximide (CHX). Mr, relative molecular mass; Ac-K, acetylated lysine.