Fig 2: PP2Ac is demethylated in the cell extracts in a time-dependent manner. HEK293T cells were lysed in a standard buffer (A,B), CHAPS buffer (C,D), and a standard buffer with OA (100 nm) (E,F). The cell extracts were incubated on ice, at 4, and 30 °C for indicated time periods, and then treated with or without NaOH (0.1 m). Demethylation of PP2Ac was detected by immunoblotting with anti-demethylated PP2Ac antibody (deM PP2Ac), and the ratio of deM PP2Ac was determined by NaOH-treated cell extracts as 100%. Representative images (A,C,E) and quantitative data (B,D,F) from three independent experiments are shown. The results are expressed as the means ± SE. VCP was used as a loading control.

Fig 3: Role of FFAs and Que on the regulation of ACACA activity. (A) HepG2 cells were incubated in low glucose (LG) or in high glucose (HG) medium, in the presence or in the absence of 0.75 mM FFAs and 5 µM Que for 24 h. Total proteins were extracted from the cells and separated by SDS/PAGE. After incubation with antibodies against phosphorylated ACACA, the protein content was quantified by densitometric analysis, and results were expressed as a percentage of control cells (LG). Results are representative of three different experiments. * p < 0.05 versus control (LG); ** p < 0.01 versus control (LG). Values are means ± S.D. (B) Cells were treated as reported in A. ACACA activity was determined in cell lysates as in Material and Methods. The activity in each sample was expressed as a percentage of control cells (LG). Values are means ± S.D., n = 3. * p < 0.05 versus control (LG); ** p < 0.01 versus control (LG). (C) After the separation of cell lysates by SDS/PAGE, proteins were transferred onto nitrocellulose membrane and blotted against phosphorylated AMPK. The protein content was quantified by densitometric analysis and expressed as a percentage of control cells (LG). Results are representative of three different experiments. * p < 0.05 versus control (LG); ** p < 0.01 versus control (LG). (D) PP2A activity was determined in cell lysates as reported in Material and Methods. The activity in each sample was expressed as a percentage of control cells (LG). Values are means ± S.D., n = 3. * p < 0.05 versus control (LG); ** p < 0.01 versus control (LG).

Fig 4: PME-1 M335D mutant has reduced binding to PP2Ac. (A) Schematic image of PME-1/PP2Ac association. (B) Recombinant His-PME-1 WT was incubated with PME-1 KO MEFs cell extracts with DMSO, ABL127 (0.1 and 1 µm), or OA (10 and 100 nm). His-PME-1 was pulled down with Ni-NTA agarose, and the association of His-PME-1 with endogenous PP2Ac was analyzed by immunoblotting. Representative images from three independent experiments are shown. (C) Recombinant His-PME-1 WT, S156A, and M335D mutants were incubated with recombinant PP2Ac, and PP2A activity was determined by in vitro PP2A activity assay. The percentage of PP2A activity was normalized by control (absence of PME-1) as 100%. N = 3 *: P < 0.05 vs control. The results are expressed as the means ± SE. Comparisons between the groups were performed by one-way analysis of variance, followed by Student–Newman–Keuls test. (D) Recombinant His-PME-1 WT, S156A, and M335D mutants were incubated with PME-1 KO MEFs cell extracts. His-PME-1 was pulled down with Ni-NTA agarose, and association of His-PME-1 with endogenous PP2Ac was analyzed by immunoblotting. Representative images from three independent experiments are shown. (E) Recombinant GST-PME-1 WT, S156A, and M335D mutants were incubated with recombinant His-PP2Ac with 5 mm EDTA, 10 mm MgCl2, or 0.2 mm MnCl2. GST-PME-1 was pulled down with Glutathione Sepharose beads, and association of GST-PME-1 with His-PP2Ac was analyzed by immunoblotting. Representative images from three independent experiments are shown.

Fig 5: A stable association of PME-1 with PP2Ac is not required for PP2Ac demethylation. (A,B) Cell extracts of PME-1 KO MEFs were incubated at 30 °C for indicated time periods with recombinant His-PME-1 WT, S156A, and M335D mutants. Demethylation of PP2Ac was analyzed by immunoblotting with anti-demethylated PP2Ac antibody (deM PP2Ac), and the ratio of deM PP2Ac was determined by NaOH-treated cell extracts as 100%. Representative images (A) and quantitative data (B) from three independent experiments are shown. The results are expressed as the means ± SE. Comparisons between the groups were performed by one-way analysis of variance, followed by Student–Newman–Keuls test. (C) Cell extracts from the PME-1 KO MEFs expressing FLAG-PME-1 WT, S156A, and M335D mutants were analyzed by immunoblotting using anti-deM PP2Ac antibody and total PP2Ac antibody. Representative images from three independent experiments are shown.