Figure 4. Effects of aging and PASK deficiency on the expression and activity of certain mitochondrial proteins and ROS/RNS content in liver. Real-time PCR was used to analyze the expression of Cox-IV (A) and Cs (B) mRNA levels. The results were measured in the livers from non-fasted (NON-FASTED) and 24-h fasted (FASTED) young (3-5 months) and aged (18-20 months) wild-type (WT) and PASK-deficient (Pask-/-) mice. Immunoblot analysis of COX-IV (C) in livers from young and aged WT and Pask-/- mice. Liver lysates from non-fasted (NF) and 24-h fasted (F) mice were processed for western blot analysis. Citrate synthase activity (D) in liver homogenates was also measured. Mitochondrial DNA content (E) and the levels of ROS/RNS (F) were also measured. The value obtained in 3-5-month-old non-fasted WT mice was taken as 1. Bar graphs in (A, B) represent the means ± SEM, and the levels of expression were normalized by the mRNA of β-actin used as housekeeping gene; (C) represents the means ± SEM, of the densitometric values normalized by total protein detected by Stain-Free (TOTAL PROTEIN) (Supplementary Figure 1); (D) represents the means ± SEM, of citrate synthase activity, expressing them as a percentage; n = 4-5 animals per condition. Bar graphs in (E, F) represent the means ± SEM. $P < 0.05, $$P < 0.01 3-5 months vs. 18-20 months; * P < 0.05, ** P < 0.01 WT vs. Pask-/-; #P < 0.05, ##P < 0.01, ###P < 0.001 non-fasted vs. fasted.