Journal: Frontiers in Veterinary Science

Article Title: The effects of valine, lysine and threonine and their optimal combinations on the synthesis of α-casein by MAC-T cells

doi: 10.3389/fvets.2026.1780614

Figure Lengend Snippet: Effects of the optimal molar ratio of valine, lysine, and threonine on the relative mRNA expression levels of downstream genes in the mTOR signaling pathway. (A–E) Represent the relative mRNA expression levels of the five key downstream targets of the mTOR pathway: EIF4EBP1 , EIF4E , S6K1 , EEF2 , and RPTOR (the detection was carried out 12 h after the amino acid supplementation treatment). The error bars represent the SD ( n = 3). The relative expression levels of genes were calculated using the 2 −ΔΔCt method, with the 0 × AA group as the calibration control group (its expression level was set to 1). Independent sample t -test was used for comparison between two groups. Different capital letters indicate extremely significant differences compared with the 0 × AA group ( p < 0.01).

Article Snippet: eEF2 , , biorbyt , orb584002 , 5% egg white , 1:1000.

Techniques: Expressing, Control, Comparison

Journal: Frontiers in Veterinary Science

Article Title: The effects of valine, lysine and threonine and their optimal combinations on the synthesis of α-casein by MAC-T cells

doi: 10.3389/fvets.2026.1780614

Figure Lengend Snippet: Effects of the optimal molar ratio of valine, lysine, and threonine on the phosphorylation levels of downstream proteins in the mTOR signaling pathway. (A) The protein phosphorylation levels of phosphorylated mTOR and S6K1 were detected by western blotting. (B) The phosphorylation level of mTOR protein (P-mTOR/mTOR) was quantified and calculated using β-actin and mTOR as internal references. (C) The phosphorylation level of S6K1 protein (P-S6K1/S6K1) was quantified and calculated using β-actin and S6K1 as internal references. (D) The protein phosphorylation levels of phosphorylated 4EBP1 and RPS6 were detected by Western blotting. (E) The phosphorylation level of 4EBP1 protein (P-4EBP1/4EBP1) was quantified and calculated using β-actin and 4EBP1 as internal references. (F) The phosphorylation level of RPS6 protein (P-RPS6/RPS6) was quantified and calculated using β-actin and RPS6 as internal references. (G) The protein phosphorylation level of phosphorylated EEF2 and the amount of α-casein were detected by Western blotting. (H) The phosphorylation level of EEF2 protein (P-EEF2/EEF2) was quantified and calculated using β-actin and EEF2 as internal references. (I) The amount of α-casein was detected by estern blotting (he detection was carried out 12 h after the amino acid supplementation treatment). The error bars represent the SD ( n = 3). Independent sample t -test was used for comparison between two groups. Different capital letters indicate extremely significant differences compared with the 0 × AA group ( p < 0.01).

Article Snippet: eEF2 , , biorbyt , orb584002 , 5% egg white , 1:1000.

Techniques: Phospho-proteomics, Western Blot, Comparison

Journal: Frontiers in Veterinary Science

Article Title: The effects of valine, lysine and threonine and their optimal combinations on the synthesis of α-casein by MAC-T cells

doi: 10.3389/fvets.2026.1780614

Figure Lengend Snippet: Inhibition of the mTOR signaling pathway to validate its role in up-regulating the expression of downstream target genes under the optimal combination of valine, lysine, and threonine. (A–E) Respectively represent the relative expression levels of key downstream protein genes of the mTOR signaling pathway (EIF4EBP1, EIF4E, S6K1, EEF2, and RPS6) in different groups (the detection was conducted 12 h after amino acid supplementation treatment). 0 × AA group: serum-free DMEM basal medium. MIX group: the optimal combination of amino acids (valine 36.114 mmol/L + lysine 9.027 mmol/L + threonine 4.602 mmol/L) was added to the basic medium. Rapamycin group: add 100 nmol/L rapamycin to the DMEM basal medium. Rapamycin+MIX group: the basic culture medium was supplemented with 100 nmol/L rapamycin and the optimal combination of amino acids simultaneously. Data were analyzed by one-way ANOVA, and the post hoc multiple comparisons were conducted using Duncan’s new multiple range test. Different lowercase letters in the figures indicate significant differences ( p < 0.05), and different uppercase letters indicate extremely significant differences ( p < 0.01).

Article Snippet: eEF2 , , biorbyt , orb584002 , 5% egg white , 1:1000.

Techniques: Inhibition, Expressing

Journal: Frontiers in Veterinary Science

Article Title: The effects of valine, lysine and threonine and their optimal combinations on the synthesis of α-casein by MAC-T cells

doi: 10.3389/fvets.2026.1780614

Figure Lengend Snippet: Inhibition of the mTOR signaling pathway to validate its role in promoting phosphorylation of downstream target proteins under the optimal combination of valine, lysine, and threonine. (A) The protein phosphorylation levels of phosphorylated mTOR and S6K1 were detected by western blotting. (B) The phosphorylation level of mTOR protein (P-mTOR/mTOR) was quantified and calculated using β-actin and mTOR as internal references. (C) The phosphorylation level of S6K1 protein (P-S6K1/S6K1) was quantified and calculated using β-actin and S6K1 as internal references. (D) The protein phosphorylation levels of phosphorylated 4EBP1 and RPS6 were detected by western blotting. (E) The phosphorylation level of 4EBP1 protein (P-4EBP1/4EBP1) was quantified and calculated using β-actin and 4EBP1 as internal references. (F) The phosphorylation level of RPS6 protein (P-RPS6/RPS6) was quantified and calculated using β-actin and RPS6 as internal references. (G) The protein phosphorylation level of phosphorylated EEF2 and the amount of α-casein were detected by western blotting. (H) The phosphorylation level of EEF2 protein (P-EEF2/EEF2) was quantified and calculated using β-actin and EEF2 as internal references. (I) The amount of α-casein was detected by western blotting (the detection was carried out 12 h after the amino acid supplementation treatment). The error bars represent the SD ( n = 3). Data were analyzed by one-way ANOVA, and the post hoc multiple comparisons were conducted using Duncan’s new multiple range test. Different lowercase letters in the figures indicate significant differences ( p < 0.05), and different uppercase letters indicate extremely significant differences ( p < 0.01).

Article Snippet: eEF2 , , biorbyt , orb584002 , 5% egg white , 1:1000.

Techniques: Inhibition, Phospho-proteomics, Western Blot