Fig. 4: Effect of NQO1 transgenesis on downstream effectors of mTORC1 and translation machinery.

a Diagram depicting the impact of excess nutrients on mTORC1 pathway activation and associated inhibition of insulin signaling. b Top panel, Skeletal muscle extracts from Fig. 3a were rerun and probed with antibodies against key downstream effectors of mTORC1, namely the phosphorylated and total forms of S6K1, ribosomal protein S6 (rpS6), 4E-BP-1, and eIF4G protein. Bottom panel, densitometric quantification of the phospho-active/total ratio of S6K1 and of 4E-BP1. Values are represented as boxplots with individual values (n = 5 per group). c Sucrose gradient fractionation of pooled cytosolic quadricep extracts from WT and NQO1-Tg mice on HFD. Fractions were resolved by SDS-PAGE and immunoblotted for rpS6 and NQO1 proteins. Monosomes are found in fractions 3–4, and polysomes in fractions 5–12. d NQO1:protein complexes in skeletal muscle homogenates were immunoprecipitated with goat anti-NQO1 antibody. Immunoprecipitates were incubated with DMSO, 10 μM beta-lapachone (β-lap.), 100 μM dicoumarol (Dic.), or 1 μg RNase A for 3 h at 4 °C, followed by immunoblot analysis (IB) with antibodies against rpl13a and NQO1. Control immunoprecipitations were carried out with magnetic beads alone and beads conjugated with goat IgG control. e Left panel, eWAT extracts from Fig. 3e were rerun and probed with anti-4E-BP1 antibody. Right panel, densitometric quantification. *, **, ***p < 0.05, 0.01, 0.001.