Figure 6. Effects of aging and PASK deficiency on NRF2, GCLm and HO1 protein expression. Immunoblot analysis of NRF2 (A), GCLm (D) and HO1 (E) in livers from young (3-5 months) and aged (18-20 months) wild-type (WT) and PASK-deficient (Pask-/-) mice. Liver lysates from non-fasted (NF) and 24-h fasted (F) mice were processed for western blot analysis. Real-time PCR was used to analyze the expression of Gclm (B), Ho1 (C) and Gclc (F) mRNAs. The value obtained in 3-5-month-old non-fasted WT mice was taken as 1. Bar graphs in (A, D, E) represent the means ± SEM of the densitometric values normalized by total protein detected by Stain-Free (TOTAL PROTEIN) (Supplementary Figure 2); (B, C, F) represent the means ± SEM, and the levels of expression were normalized by the mRNA of β-actin, n = 4-5 animals per condition. $P < 0.05, $$P < 0.01 3-5 months vs. 18-20 months; * P < 0.05, ** P < 0.01, *** P < 0.001 WT vs. Pask-/-; #P < 0.05, ##P < 0.01 non-fasted vs. fasted.