Journal: Biology

Article Title: Selection and Evaluation of mRNA and miRNA Reference Genes for Expression Studies (qPCR) in Archived Formalin-Fixed and Paraffin-Embedded (FFPE) Colon Samples of DSS-Induced Colitis Mouse Model

doi: 10.3390/biology12020190

Figure Lengend Snippet: Information on mRNA candidate reference genes and target genes and their qPCR amplification characteristics in archived FFPE colon samples of control and DSS-treated C57BL6/JOlaHsd mice. Legend: Eff., amplification efficiency; R2, correlation coefficient.

Article Snippet: EEF2 , Eucaryotic translation elongation factor 2 , Mm01171434_g1 , 74 , 4331182 , 95 , 0.998 , 102 , 0.997.

Techniques: Amplification, Control, Binding Assay

Journal: Biology

Article Title: Selection and Evaluation of mRNA and miRNA Reference Genes for Expression Studies (qPCR) in Archived Formalin-Fixed and Paraffin-Embedded (FFPE) Colon Samples of DSS-Induced Colitis Mouse Model

doi: 10.3390/biology12020190

Figure Lengend Snippet: Basic statistics of the Cq-values of candidate reference genes in archived FFPE colon samples of control and DSS-treated C57BL6/JOlaHsd mice (n=117). Legend: SD, standard deviation; TBP+, 16 of 117 FFPE samples were undetermined for TBP, therefore, only 101 FFPE samples in which TBP was expressed were included in the calculation.

Article Snippet: EEF2 , Eucaryotic translation elongation factor 2 , Mm01171434_g1 , 74 , 4331182 , 95 , 0.998 , 102 , 0.997.

Techniques: Control, Standard Deviation

Journal: Biology

Article Title: Selection and Evaluation of mRNA and miRNA Reference Genes for Expression Studies (qPCR) in Archived Formalin-Fixed and Paraffin-Embedded (FFPE) Colon Samples of DSS-Induced Colitis Mouse Model

doi: 10.3390/biology12020190

Figure Lengend Snippet: Expression stability of candidate reference genes in archived FFPE colon samples of control and DSS-treated C57BL6/JOlaHsd mice, calculated with BestKeeper. Legend: F, female; M, male; CP, crossing point (BestKeeper’s notation for Cq); SD, standard deviation; grey cells denote control groups; white cells denote DSS groups; red cells denote low expression stability (high SD-values > 1); TBP+, number of samples (n) included in the calculation of TBP is smaller than shown in the first column (16 of 117 FFPE samples were undetermined for TBP, 2 from the control and 14 from DSS group, all undetermined FFPE samples were males).

Article Snippet: EEF2 , Eucaryotic translation elongation factor 2 , Mm01171434_g1 , 74 , 4331182 , 95 , 0.998 , 102 , 0.997.

Techniques: Expressing, Control, Standard Deviation

Journal: Biology

Article Title: Selection and Evaluation of mRNA and miRNA Reference Genes for Expression Studies (qPCR) in Archived Formalin-Fixed and Paraffin-Embedded (FFPE) Colon Samples of DSS-Induced Colitis Mouse Model

doi: 10.3390/biology12020190

Figure Lengend Snippet: Selection of reference genes affects relative expression levels of target genes and the impact of inter/intra-experimental factors on target gene expression in archived FFPE samples of control and DSS-treated C57BL6/JOlaHsd mice (females and males). ( a, b ) Graphs present relative expression level of ( a ) TNFR1 and TNFR2, ( b ) miR-181a-5p and miR-223p ( a ) normalized to the most (EEF2/RPLP0) and to the least stable (PPIA and NONO) mRNA reference genes and ( b ) to the most (miR-191-5p/miR-103a-3p/miR-16-5p) and to the least stable (U6) miRNA reference genes, respectively. Different normalization led to significant differences. ( c ) Graphs represent influence of inter/intra-experimental factors on absolute (no normalization) and relative expression levels of TNFR1 (normalized to the combination of reference genes EEF2/RPLP0, and PPIA and NONO). Normalization of TNFR1 reduced the influence of inter/intra-experimental factors on relative expression levels of TNFR1 (factors inside . The number of inter/intra-experimental factors that do not affect TNFR1 is greater in the case of normalization with the most stable mRNA reference genes (EEF2/RPLP0). Legend: F, female; M, male; E1–E9, experiment number; DSS, DSS treatment applied; duration of inflammation, days/months of water after DSS treatment; Hy, histology; normal, no lesion or inflammation; mild, moderate and severe refer to inflammation; erosion, destruction of epithelial layer of mucosa; heterogeneous, includes both erosion and inflammation. Statistical significance: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: EEF2 , Eucaryotic translation elongation factor 2 , Mm01171434_g1 , 74 , 4331182 , 95 , 0.998 , 102 , 0.997.

Techniques: Selection, Expressing, Targeted Gene Expression, Control

Journal: Science advances

Article Title: The autism susceptibility kinase, TAOK2, phosphorylates eEF2 and modulates translation.

doi: 10.1126/sciadv.adf7001

Figure Lengend Snippet: Fig. 3. TAOK2β binds eEF2 and represses translation through direct phosphorylation of its Thr56 residue. (A) Volcano plot of Taok2 knockout/Taok2 WT phospho- proteome (left, prefrontal cortex; middle, hippocampus; right, ventral striatum). Phosphopeptides containing the TAOK2 kinase-recognition motif pTXX[K/R/H] are in purple and highlight potential direct substrates of TAOK2. Two data points are outside of axis limits. (B and C) Immunoblot and quantifications of phosphorylated eEF2 (Thr56) from cortical lysates from Taok2+/+ and Taok2−/− mice (n = 5); *P < 0.05, SEM error bars, unpaired t test. β-Actin was used as a loading control. (D) Immunoblot of the TAOK2β IP from nontransfected N2a cells and N2a cells overexpressing WT TAOK2β shows the interaction of TAOK2β with eEF2. (E) Immunoblots of in vitro kinase as- says using recombinant proteins as depicted in the figure show the direct phosphorylation of eEF2 (Thr56) by TAOK2 kinase. (F and G) Immunoblot and quantifications of phospho-eEF2 from N2a cell lysates transfected with WT TAOK2β and its respective control (cells expressing pcDNA3.1-myc-tag). n = 3 per condition from different inde- pendent transfection experiments; *P < 0.05, SEM error bars, unpaired t test. (H and I) Immunoblot and quantifications of phospho-eEF2 from LCLs lysates with TAOK2 mutation A135P (patient) and the nonaffected father. n = 3 per condition; **P < 0.05; SEM error bars, unpaired t test. (J and K) Immunoblot and quantifications of phospho-eEF2 from N2a cell lysates transfected with TAOK2βA135P and its respective control (cells expressing pcDNA3.1-myc-tag). n = 4 per condition from different independent transfection experiments; SEM error bars, **P < 0.01 by unpaired t test. β-Actin was used as a loading control.

Article Snippet: Either ascending concentrations of 25, 50, 100, and 150 ng of recombinant human protein TAOK2 (amino acids 1 to 314) expressed by baculovirus in Sf9 insect cells using N- terminal GST tag (Signal- Chem, T25- 11G- 10), or 150 ng of TAOK2 protein (amino acids 1 to 314) containing 60 μm of TAOK2 inhibitor compound 43 (Cp 43) (Tocris, 6558) or equivalent concentration of vehicle dimethyl sulfoxide (DMSO), or 250 ng of TAOK2, recombinant protein 1- 314 with mutation A135P, expressed by baculovirus in Sf9 insect cells using N- terminal GST tag (Origene, SR248141; custom made), or its control TAOK2 (amino acids 1 to 314) was incubated with 300 ng of recombinant human protein eEF2 with N- terminal 6× His- tag (MyBioSource, MBS1213669) or recombinant human protein eEF2 with mutation in T56A expressed in Escherichia coli with N- terminal 6× His- tag (Origene, SR248140; custom made) in a 25- μl kinase assay buffer I (Signal- Chem, K01- 09) supplemented with cOmplete protease inhibitor cocktail (Roche) and phosSTOP (Roche).

Techniques: Phospho-proteomics, Residue, Knock-Out, Western Blot, Control, In Vitro, Recombinant, Transfection, Expressing, Mutagenesis

Journal: Science advances

Article Title: The autism susceptibility kinase, TAOK2, phosphorylates eEF2 and modulates translation.

doi: 10.1126/sciadv.adf7001

Figure Lengend Snippet: Fig. 3. TAOK2β binds eEF2 and represses translation through direct phosphorylation of its Thr56 residue. (A) Volcano plot of Taok2 knockout/Taok2 WT phospho- proteome (left, prefrontal cortex; middle, hippocampus; right, ventral striatum). Phosphopeptides containing the TAOK2 kinase-recognition motif pTXX[K/R/H] are in purple and highlight potential direct substrates of TAOK2. Two data points are outside of axis limits. (B and C) Immunoblot and quantifications of phosphorylated eEF2 (Thr56) from cortical lysates from Taok2+/+ and Taok2−/− mice (n = 5); *P < 0.05, SEM error bars, unpaired t test. β-Actin was used as a loading control. (D) Immunoblot of the TAOK2β IP from nontransfected N2a cells and N2a cells overexpressing WT TAOK2β shows the interaction of TAOK2β with eEF2. (E) Immunoblots of in vitro kinase as- says using recombinant proteins as depicted in the figure show the direct phosphorylation of eEF2 (Thr56) by TAOK2 kinase. (F and G) Immunoblot and quantifications of phospho-eEF2 from N2a cell lysates transfected with WT TAOK2β and its respective control (cells expressing pcDNA3.1-myc-tag). n = 3 per condition from different inde- pendent transfection experiments; *P < 0.05, SEM error bars, unpaired t test. (H and I) Immunoblot and quantifications of phospho-eEF2 from LCLs lysates with TAOK2 mutation A135P (patient) and the nonaffected father. n = 3 per condition; **P < 0.05; SEM error bars, unpaired t test. (J and K) Immunoblot and quantifications of phospho-eEF2 from N2a cell lysates transfected with TAOK2βA135P and its respective control (cells expressing pcDNA3.1-myc-tag). n = 4 per condition from different independent transfection experiments; SEM error bars, **P < 0.01 by unpaired t test. β-Actin was used as a loading control.

Article Snippet: Either ascending concentrations of 25, 50, 100, and 150 ng of recombinant human protein TAOK2 (amino acids 1 to 314) expressed by baculovirus in Sf9 insect cells using N- terminal GST tag (Signal- Chem, T25- 11G- 10), or 150 ng of TAOK2 protein (amino acids 1 to 314) containing 60 μm of TAOK2 inhibitor compound 43 (Cp 43) (Tocris, 6558) or equivalent concentration of vehicle dimethyl sulfoxide (DMSO), or 250 ng of TAOK2, recombinant protein 1- 314 with mutation A135P, expressed by baculovirus in Sf9 insect cells using N- terminal GST tag (Origene, SR248141; custom made), or its control TAOK2 (amino acids 1 to 314) was incubated with 300 ng of recombinant human protein eEF2 with N- terminal 6× His- tag (MyBioSource, MBS1213669) or recombinant human protein eEF2 with mutation in T56A expressed in Escherichia coli with N- terminal 6× His- tag (Origene, SR248140; custom made) in a 25- μl kinase assay buffer I (Signal- Chem, K01- 09) supplemented with cOmplete protease inhibitor cocktail (Roche) and phosSTOP (Roche).

Techniques: Phospho-proteomics, Residue, Knock-Out, Western Blot, Control, In Vitro, Recombinant, Transfection, Expressing, Mutagenesis

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