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. 2023 Aug 17;14:4987. doi: 10.1038/s41467-023-40597-z

Fig. 1. PAK4 protein levels are repressed by fasting.

Fig. 1

a Eight-week-old male C57BL/6 mice were either fed a normal chow diet (NCD) ad libitum, fasted for 6–36 h, or refed for 1 or 3 h following a 36 h fast. Liver tissues were subjected to Western blotting to determine PAK4 protein levels (n = 3). **P < 0.01 versus fed; #P < 0.05 versus fasted for 36 h. b Eight-week-old male C57BL/6 mice were fed either a NCD or a ketogenic diet (KD) for 2 weeks, and hepatic PAK4 protein levels were analyzed. c Representative immunoblot images for PAK4 expression in liver tissue obtained from mice fed either a NCD or a 60% high-fat diet (HFD) for 16 weeks or from ob/ob or db/db mice and their control groups. d AML12 cells transfected with HA-Ub and PAK4 were treated with glucagon (GCG, 100 nM) for 12 h in the presence or absence of H89 (10 μM). Cell lysates were immunoprecipitated with anti-PAK4 antibody and immunoblotted with anti-ubiquitin (Ub) antibody. e, f Mouse primary hepatocytes were treated with glucagon (100 nM) with or without H89 (10 μM), or octanoate (OCA, 2 mM) with or without EX-527 (100 nM). Subsequently, the cells were treated with cycloheximide (CHX, 100 μg/ml) for the indicated time periods. The protein levels of PAK4 were compared (n = 3). *P < 0.05 versus vehicle (Veh); #P < 0.05 and ##P < 0.01 versus GCG or OCA. g Primary hepatocytes were treated with octanoate (2 mM) for 12 h in the presence or absence of H89 (10 μM) or EX-527 (100 nM). Cell lysates were immunoprecipitated with anti-PAK4 antibody and immunoblotted with anti-ubiquitin (Ub) antibody. h, i AML12 cells were transfected with wild-type (WT) or mutant PAK4 (S181A, S258A, or S474A) and then treated with forskolin (Fsk, 10 μM) for 12 h to compare protein degradation and ubiquitination of PAK4. j AML12 cells transfected with either control siRNA or siRNA targeting MDM2 were treated with glucagon (100 nM) for 12 h. Cell lysates were immunoblotted for indicated proteins or immunoprecipitated with anti-PAK4 antibody followed by immunoblotting with anti-ubiquitin (Ub) antibody. k Schematic summary of PKA- and Sirt1-mediated PAK4 degradation. Data are presented as the mean ± SEM. One-way ANOVA followed by Dunnett’s multiple comparisons test (a) and Tukey’s multiple comparisons test (e, f) were conducted for statistical analyses. Source data are provided as a Source Data file.