Figure S4.
Tip60 regulates mTORC1 activity and insulin sensitivity through acetylating Rheb at lysine 8 (K8) under PA treatment. (A and B) Immunoblotting of Raptor acetylation in fish myocytes (A) and C2C12 myotubes (B) with the indicated concentrations of PA for 12 h (n = 3 independent wells per treatment). (C) Representative MS/MS spectrum of acetylated K97, K102, K161, or K169 peptides of Rheb in mouse. (D) Sequence alignment of Rheb from the human, mouse, and large yellow croaker. The red box indicated the conserved lysine 8 (K8) and the blue boxes indicated the conserved lysine 5 (K5), lysine 19 (K19), lysine 45 (K45), lysine 91 (K91), lysine 102 (K102), lysine 109 (K109), lysine 120 (K120), lysine 135 (K135), lysine 151 (K151), lysine 169 (K169), and lysine 178 (K178). (E) The activity of mTORC1 signaling was analyzed by immunoblotting in fish myocytes transfected with the indicated plasmids. (F) The activity of mTORC1 signaling was measured by immunoblotting in HEK293T cells transfected with the indicated plasmids. (G) The activity of mTORC1 signaling was measured by immunoblotting in C2C12 myotubes treated with the indicated concentrations of CBP/P300 inhibitor C646 under control or 500 μM PA treatment for 12 h (n = 3 independent wells per treatment). (H) The activity of mTORC1 signaling was tested by immunoblotting in C2C12 myotubes treated with the indicated concentrations of CBP/P300 inhibitor spermidine under control or 500 μM PA treatment for 12 h (n = 3 independent wells per treatment). (I) The activity of mTORC1 signaling was tested by immunoblotting in C2C12 myotubes treated with the indicated concentrations of GCN5 inhibitor MB-3 under control or 500 μM PA treatment for 12 h (n = 3 independent wells per treatment). (J and K) Relative mRNA (J) and protein (K) levels of Tip60 were analyzed by quantitative PCR and immunoblotting in C2C12 myotubes transfected with control siRNA or siRNA against Tip60 for 48 h (n = 3 independent wells per treatment). (L and M) Relative mRNA levels of tip60 were analyzed by quantitative PCR in fish myocytes under the indicated concentrations of OA (L) or LA (M) treatment for 12 h (n = 3 independent wells per treatment). The results are presented as the mean ± SEM and were analyzed using independent t tests (*P < 0.05, **P < 0.01). Source data are available for this figure: SourceData FS4.

Tip60 regulates mTORC1 activity and insulin sensitivity through acetylating Rheb at lysine 8 (K8) under PA treatment. (A and B) Immunoblotting of Raptor acetylation in fish myocytes (A) and C2C12 myotubes (B) with the indicated concentrations of PA for 12 h (n = 3 independent wells per treatment). (C) Representative MS/MS spectrum of acetylated K97, K102, K161, or K169 peptides of Rheb in mouse. (D) Sequence alignment of Rheb from the human, mouse, and large yellow croaker. The red box indicated the conserved lysine 8 (K8) and the blue boxes indicated the conserved lysine 5 (K5), lysine 19 (K19), lysine 45 (K45), lysine 91 (K91), lysine 102 (K102), lysine 109 (K109), lysine 120 (K120), lysine 135 (K135), lysine 151 (K151), lysine 169 (K169), and lysine 178 (K178). (E) The activity of mTORC1 signaling was analyzed by immunoblotting in fish myocytes transfected with the indicated plasmids. (F) The activity of mTORC1 signaling was measured by immunoblotting in HEK293T cells transfected with the indicated plasmids. (G) The activity of mTORC1 signaling was measured by immunoblotting in C2C12 myotubes treated with the indicated concentrations of CBP/P300 inhibitor C646 under control or 500 μM PA treatment for 12 h (n = 3 independent wells per treatment). (H) The activity of mTORC1 signaling was tested by immunoblotting in C2C12 myotubes treated with the indicated concentrations of CBP/P300 inhibitor spermidine under control or 500 μM PA treatment for 12 h (n = 3 independent wells per treatment). (I) The activity of mTORC1 signaling was tested by immunoblotting in C2C12 myotubes treated with the indicated concentrations of GCN5 inhibitor MB-3 under control or 500 μM PA treatment for 12 h (n = 3 independent wells per treatment). (J and K) Relative mRNA (J) and protein (K) levels of Tip60 were analyzed by quantitative PCR and immunoblotting in C2C12 myotubes transfected with control siRNA or siRNA against Tip60 for 48 h (n = 3 independent wells per treatment). (L and M) Relative mRNA levels of tip60 were analyzed by quantitative PCR in fish myocytes under the indicated concentrations of OA (L) or LA (M) treatment for 12 h (n = 3 independent wells per treatment). The results are presented as the mean ± SEM and were analyzed using independent t tests (*P < 0.05, **P < 0.01). Source data are available for this figure: SourceData FS4.

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