Fig 2: Milk protein synthesis mTOR related signaling pathway protein expressions. (A) mTOR related signaling pathway protein expression levels were measured by western blotting analysis. (B) The ratios of phosphorylated to total mTOR were quantified by gray scale scan. (C) The ratios of phosphorylated P70S6K to total P70S6K were quantified by gray scale scan. (D) The ratios of phosphorylated eIF4E to total eIF4E were quantified by gray scale scan. (E) The ratios of phosphorylated eEF2K to total eEF2K were quantified by gray scale scan. (F) Relative protein levels of eEF2 from the western blots were quantified by gray scale scan. Values are mean ± SEM. n = 4/group. bp < 0.05 and ap < 0.01, compared with high grain group.

Fig 3: Effects of high-grain diet with buffering agent on the milk protein synthesis. Feeding of high-grain diet with buffering agent promoted the jugular vein blood of amino acids concentration, and the mRNA expressions of amino acid transport SLC1A3, SLC1A5, SLC7A5, SLC7A6, and SLC38A2 in mammary tissues were up-regulated, it showed that more amino acids flowed into the mammary. Amino acids in mammary gland activated mTOR pathway. mTORC1 activates P70S6K via phosphorylation, which in turn activated several proteins that contributed to promote protein synthesis. In addition, activation of P70S6K inhibited eEF2K via phosphorylation, subsequently stimulated eEF2, which activated translocation elongation. When mTORC1 was phosphorylated, it inhibited 4EBP1 through phosphorylation, which inhibits eIF4E. eIF4E activates cap-dependent translocation increasing protein synthesis. The blue arrow represents promotion, and the red arrow represents inhibition.

Fig 4: p-eEF2K Ser366 physically interacts with MEK1/2. (A) Co-IP assay of proteins MEK1/2 and p-eEF2K Ser366 in MKN45 cells. Representative co-IP immunoblot (upper) and quantification of the relative gray intensities (lower). IgG was used as a control. The relative gray intensities represent the ratios of p-eEF2K Ser366 between the co-IP of anti-MEK1/2/ERK1/2 or IgG and the co-IP of CK (control condition) were calculated and normalized to the IP in CK (n=3). *P<0.05 vs. IgG. NS, not significant. (B) Immunofluorescence confocal microscopy of eEF2K and MEK1/2 in MKN45 cells grown in complete medium or under nutrition deprivation for 3 h. The cells were incubated with anti-eEF2K or anti-MEK1/2 and with fluorescent secondary antibodies conjugated with AlexaFluor 555 (red) and 488 (green) for eEF2K and MEK1/2, respectively. Scale bar, 25 µm. eEF2K, eukaryotic elongation factor 2 kinase; MEK1/2, dual-specificity mitogen-activated protein kinase kinase 1/2; IP, immunoprecipitation; CK, cell lysate; IgG, immunoglobulin G.

Fig 5: Knockdown of eEF2K reduced the activity of ERK1/2. Cells were transiently transfected with 25 nM siCTRL or sieEF2K, cultured for 48 h, treated and analyzed by western blotting. (A) The effect of eEF2K knockdown on ERK1/2 activity in complete medium. Representative immunoblot (left) and quantification of the relative gray intensities of p-ERK1/2 T202/Y204 normalized to ERK1/2 (right). *P<0.05 vs. siCTRL. (B) The effect of ND treatment and eEF2K knockdown on ERK1/2 activity. The cells were treated under ND for 3 h and analyzed by western blotting. Representative immunoblot (left) and quantification of the relative gray intensities of p-ERK1/2 T202/T204 normalized to ERK1/2 (right). *P<0.05. vs. ND + siCTRL. eEF2K, eukaryotic elongation factor 2 kinase; ERK1/2, mitogen-activated protein kinase; P-ERK1/2, phosphorylated ERK1/2; siCTRL, siRNA control; sieEF2K, eEF2K siRNA; ND, nutrition deprivation.