Levels of HSL protein, HSL phosphorylation, DG and of the DG/(TG + DG) ratio, and ATGL protein in WAT of 5-h and 24-h fasted mice. (A) Levels of HSL and HSL phosphorylation. Protein was extracted from WAT of wild type mice. Expression levels of total HSL, and of two phosphorylated forms of HSL, HSL-Ser563, and HSL-Ser660, were measured by Western blotting. The bar graphs show the quantification of blots from four different mice (mean ± SEM) by image J software. In each case, the level of HSL at 5 h of fasting is assigned a value of unity and the level at 24 h of fasting is expressed relative to it. (B) Measurement of DG content and (C) of the DG/(TG + DG) ratio of mouse WAT after 5 h or 24 h of fasting. (D) Levels of ATGL protein in the lipid droplet and cytosolic fractions of HSL flox/flox and HSLAKO mice after 5 h or 24 h of fasting were detected by Western blotting. Results were quantified from three different mice in each group. Values are mean ± S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001.