Journal: Science Advances

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

doi: 10.1126/sciadv.adf7001

Figure Lengend Snippet: ( A ) Volcano plot of Taok2 knockout/ Taok2 WT phosphoproteome (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 (Thr 56 ) 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 assays using recombinant proteins as depicted in the figure show the direct phosphorylation of eEF2 (Thr 56 ) 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 independent 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: One microgram from the recombinant human protein TAOK2 (amino acids 1 to 314) (Signal-Chem, T25-11G-10) or the recombinant human protein eEF2 (MyBioSource, MBS1213669) was subjected to process of denaturation, reduction, alkylation, and digestion and processed for normal proteomics as mentioned previously.

Techniques: Knock-Out, Western Blot, In Vitro, Recombinant, Transfection, Expressing, Mutagenesis

Journal: PLoS ONE

Article Title: Hypoxia Integration in the Serological Proteome Analysis Unmasks Tumor Antigens and Fosters the Identification of Anti-Phospho-eEF2 Antibodies as Potential Cancer Biomarkers

doi: 10.1371/journal.pone.0076508

Figure Lengend Snippet: A. Representative immunoblotting of 2D-separated lysates of hypoxic HCT116 cells with a commercial antibody against eEF2. Proteins of the lysates are labelled with Cy dye (red) and secondary antibody is conjugated to horseradish peroxidase (green spots). Positive signal is obtained for several spots of the same molecular weight but differing by their pI value. B. Comparison of the eEF2 spots detected with a commercial antibody against total eEF2 (top), the serum from tumor-bearing mice (middle) and a commercial antibody against phospho-Thr56 eEF2 (bottom). Spot 4 (rightmost spot) corresponds to the unphosphorylated form of eEF2 while the other spots correspond to multi-phosphorylated forms of the protein; spot 3 (second spot from the right) corresponds to the preferential monophosphorylated form of eEF2 (on Thr56).

Article Snippet: The phosphopeptide sequence was RAGETRFTDTRK, corresponding to amino acids 50 to 61 of the eEF2 protein, with a phosphorylation on Thr56 (Eurogentec).

Techniques: Western Blot, Molecular Weight

Journal: PLoS ONE

Article Title: Hypoxia Integration in the Serological Proteome Analysis Unmasks Tumor Antigens and Fosters the Identification of Anti-Phospho-eEF2 Antibodies as Potential Cancer Biomarkers

doi: 10.1371/journal.pone.0076508

Figure Lengend Snippet: A. Representative eEF2 and phospho-Thr56 eEF2 immunoblotting of HCT116 and HT29 cultured for 48 hours under hypoxia (Hx) or maintained in normoxia (Nx). B. Normalized expression of phospho-Thr56 eEF2 in normoxic vs hypoxic HCT116 and HT29 cells; n = 3, **p<0.01 C. Representative phospho-Thr56 eEF2 immunostaining of sections of HCT116 tumors in the absence (top) or the presence (bottom) of phosphatase lambda; note the complete disappearance of the phosphorylated form of eEF2 upon treatment with the phosphatase.

Article Snippet: The phosphopeptide sequence was RAGETRFTDTRK, corresponding to amino acids 50 to 61 of the eEF2 protein, with a phosphorylation on Thr56 (Eurogentec).

Techniques: Western Blot, Cell Culture, Expressing, Immunostaining

Journal: PLoS ONE

Article Title: Hypoxia Integration in the Serological Proteome Analysis Unmasks Tumor Antigens and Fosters the Identification of Anti-Phospho-eEF2 Antibodies as Potential Cancer Biomarkers

doi: 10.1371/journal.pone.0076508

Figure Lengend Snippet: A. Human and mouse amino acid sequences of eEF2 in the region of Thr56. The 12 residues corresponding to the synthetic peptide (phosphorylated on Thr56) used in our immunoassay are indicated (red frame); note the perfect identity between mouse and human sequences. B. Detection of commercial anti-phospho-Thr56 eEF2-antibodies using our immunoassay; dashed lines show the 95% confidence band of the linear regression. C. Detection of anti-phospho-Thr56 eEF2 aAb in the serum of control or HCT116 tumor-bearing mice (n = 3). ***P<0.001. D. Time course of HCT116 tumor growth as determined by measurements of tumor diameters (n = 7 per group). E. Detection of anti-phospho-Thr56 eEF2 aAb at the indicated time of HCT116 tumor progression. *P<0.05, **P<0.01, ***P<0.001 (n = 6–7 per group). Note that at day 7 post-implantation, tumors are not detectable (see panel D) but a positive signal is detected in the immunoassay. F. Graph represents the detection of anti-phospho-Thr56 eEF2 aAb in the serum of control subjects (n = 6) and patients with adenomatous polyps (n = 14) or carcinoma (n = 9). *P<0.05, **P<0.01. Of note, K-means clustering identified two subpopulations of patients (see black and red symbols) among individuals diagnosed with adenomatous polyps (P<0.001) and carcinoma (P<0.01); the same partition was observed in 100 independent runs by varying the random initialization of K-means algorithm.

Article Snippet: The phosphopeptide sequence was RAGETRFTDTRK, corresponding to amino acids 50 to 61 of the eEF2 protein, with a phosphorylation on Thr56 (Eurogentec).

Techniques: