Lack of AMPK-ACC phosphorylation does not affect AMPK signaling or phosphorylation of cytoskeletal proteins. (A) Washed murine and human platelets were lysed and subjected to western blotting for ACC1 or ACC2 isoform expression analysis. Isolated rat cardiomyocytes (CM) and mouse liver extracts served as positive controls for the detection of ACC2 and ACC1, respectively. (B-E) ACC WT and ACC DKI platelets were stimulated with 0.2 U/mL thrombin (B,D) or 5 µg/mL collagen (C,E) for the indicated times. Whole-platelet lysates were subjected to western blotting and probed with Ser79 phosphorylated ACC Ab (B-C), Thr172 phosphorylated AMPK, Ser19 phosphorylated myosin light chain (MLC), Ser3 phosphorylated cofilin and Thr278 phosphorylated vasodilator-stimulated phosphoprotein (VASP) Abs (D-E). Gelsolin and β-actin were used as loading controls. Quantification and representative western blotting are systematically shown. The solid lines on the western blots indicate that samples were run on the same gel but were not contiguous. The results are expressed as means ± standard error of the mean (SEM; at least 3 experiments for each condition). *Values statistically different from respective untreated platelets; P ≤ .05. Analysis was performed by 2-way analysis of variance (ANOVA). See also supplemental Figure 1 and supplemental Table 1.