Journal: Cell Systems

Article Title: Context Specificity in Causal Signaling Networks Revealed by Phosphoprotein Profiling

doi: 10.1016/j.cels.2016.11.013

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-eEF2 , Cell Signaling Technology , Cat#2332; RRID: AB_10693546.

Techniques: Transduction, Recombinant, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Software, Inhibition, Modification

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: The Journal of comparative neurology

Article Title: Proteomic analyses of nucleus laminaris identified candidate targets of the fragile X mental retardation protein

doi: 10.1002/cne.24281

Figure Lengend Snippet: Primary antibodies used for immunocytochemistry and other staining. Abbreviations: ICC: immunocytochemistry; WB: Western blot.

Article Snippet: Specific secondary HRP-conjugated antibodies were used at 1:2500 dilution (Santa Cruz, Biotechonology®, Inc., Dallas, TX) and blots were developed with SuperSignal™ West Pico Chemiluminescent Substrate (Thermo Scientific, Inc., Waltham, MA) and exposed to X-ray film. table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Immunogen Manufacturer, catalog number, Host, monoclonal or polyclonal; RRID, Concentration Anti-MAP2 Bovine brain MAP2 (aa 997–1332) Millipore (Billerica, MA), MAB3418; Mouse monoclonal; RRID:AB_94856 1:1000 (ICC); 1:1000 (WB) Anti-MAP2 Synthetic peptide rat MAP2 (aa 1-100) Abcam (Cambridge, MA), ab32454; Rabbit polyclonal; RRID:AB_776174 1:1000 (ICC) Anti-MAP1B Full length rat brain MAP1B Abcam, Ab11266; Mouse monoclonal IgG1 clone AA6; RRID:AB_297884 1:10,000 (ICC); 1:1000 (WB) Anti-TuJ-1 Rat brain tubulin beta-3 R&D Systems (Minneapolis, MN), MAB1195; Mouse monoclonal IgG2a; RRID:AB_357520 1:10,000 (ICC); 1:1000 (WB) Anti-eEF1a Crude calmodulin-binding proteins from Trypanosoma brucei chromatography Millipore, 05-235; Mouse monoclonal IgG1k clone CBP-KK1; RRID:AB_309663 1:1000 (ICC); 1:1000 (WB) Anti-p-eEF2 Synthetic phosphopeptide surrounding Thr56 of human eEF2, GETRFtDTRK Cell Signaling Technology (Danvers, MA), No. 2331; Rabbit polyclonal; RRID:AB_2277755 1:1000 (ICC) Anti-NSF-1 Recombinant human full length NSF.

Techniques: Immunocytochemistry, Staining, Western Blot, Concentration Assay, Chromatography, Recombinant, Purification

Journal: The Journal of Biological Chemistry

Article Title: The FAM86 domain of FAM86A confers substrate specificity to promote EEF2-Lys525 methylation

doi: 10.1016/j.jbc.2023.104842

Figure Lengend Snippet: The FAM86 domain is required for EEF2 methylation by FAM86A. A , Western blot analysis of whole cell extracts from control or FAM86A-depleted HEK293T cells. B , schematic of wildtype FAM86A and FAM86A(101–330) lacking the FAM86 domain. C , Western blot analysis of whole cell extracts from FAM86A-depleted HEK293T cells complemented with the indicated constructs described in ( B ). D , in vitro methylation assay with recombinant wildtype or truncated FAM86A as indicated with recombinant EEF2 as substrate. Top panel, 3 H-SAM is the methyl donor and methylation visualized by autoradiography. Bottom panel, Coomassie blue stain of the proteins in the reaction. E , in vitro methylation assay as in ( D ) using whole cell extracts from FAM86A-depleted HEK293T cells shown in ( A ) as the substrate.

Article Snippet: For protein expression and purification from human cells, EEF2 (Uniprot ID: P13639 ) and its mutants were cloned into pcDNA3.1(+) (Addgene) including the N-terminal FLAG sequence DYKDDDDK.

Techniques: Methylation, Western Blot, Control, Construct, In Vitro, Recombinant, Autoradiography, Staining

Journal: The Journal of Biological Chemistry

Article Title: The FAM86 domain of FAM86A confers substrate specificity to promote EEF2-Lys525 methylation

doi: 10.1016/j.jbc.2023.104842

Figure Lengend Snippet: Simulation of the FAM86A–EEF2 interface by AlphaFold-Multimer. A , ribbon representation of the overall complex predicted by AlphaFold-Multimer. EEF2 is colored in green . FAM86A is colored in blue for the MTase domain and pink for the FAM86 domain. S- adenosyl methionine (SAM) is shown as spheres. Spaces containing intermolecular interaction within 3 Å are designated as (a) near the FAM86A active site and (b) for the interaction involving the FAM86 domain of FAM86A and domain IV of EEF2. B , close-up view of (a) showing the proximity of EEF2-Lys525 to the SAM-binding pocket of FAM86A. C , close-up view of (b) showing the intermolecular interaction between the FAM86 domain of FAM86A and domain IV of EEF2. D and E , overlays of FAM86A ( D ) and EEF2 ( E ) individual AlphaFold models superimposed onto the FAM86A–EEF2 complex from AlphaFold-Multimer. Models of FAM86A and EEF2 determined individually are shown in blue , and models determined in complex with one another are shown in orange .

Article Snippet: For protein expression and purification from human cells, EEF2 (Uniprot ID: P13639 ) and its mutants were cloned into pcDNA3.1(+) (Addgene) including the N-terminal FLAG sequence DYKDDDDK.

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: The FAM86 domain of FAM86A confers substrate specificity to promote EEF2-Lys525 methylation

doi: 10.1016/j.jbc.2023.104842

Figure Lengend Snippet: Allosteric inhibition of EEF2 methylation by point mutation of FAM86A and EEF2. A , Western blot analysis of whole cell extracts from FAM86A-depleted HEK293T cells complement with either wildtype or A97R-substituted FAM86A. B , FLAG immunoprecipitation of either wildtype or I722R-substituted EEF2 from HEK293T cells. C and D , in vitro methylation assays as in Figure 2 with the indicated combinations.

Article Snippet: For protein expression and purification from human cells, EEF2 (Uniprot ID: P13639 ) and its mutants were cloned into pcDNA3.1(+) (Addgene) including the N-terminal FLAG sequence DYKDDDDK.

Techniques: Inhibition, Methylation, Mutagenesis, Western Blot, Immunoprecipitation, In Vitro

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: PLoS ONE

Article Title: Inhibition of Elongation Factor-2 Kinase Augments the Antitumor Activity of Temozolomide against Glioma

doi: 10.1371/journal.pone.0081345

Figure Lengend Snippet: ( A ) Human glioma cell lines LN229 and U251 with or without silencing of eEF-2 kinase expression were plated in 96-well plates (5×10 3 /well) and treated with TMZ (100 μM) at 37°C for 24 h; ( B ) LN229 and U251 cells were plated in 96-well plates (5×10 3 /well) and treated with TMZ (100 μM) in the presence or absence of 0.5μM of NH125 at 37°C for 24 h. Cell viability was measured by CCK-8 assay. ( C ) LN229 and U251 cells were transfected with an eEF-2 kinase-targeted siRNA or a non-targeting control siRNA (NT control). At different time points, expression of eEF-2 kinase was analyzed by Western blot,. β-actin was used as a loading control. ( D ) LN229 and U251 cells were treated with NH125 (0.5μM) or vehicle for various periods of time. At the end of treatment, the level of phospho-eEF2 (Thr56) was examined by western blot. β-actin was used as a loading control. Each bar represents mean ± SD of triplicate determinations; results shown are the representative of three identical experiments; data are expressed as the percentage of the controls. ** p < 0.01.

Article Snippet: Temozolomide and dimethyl sulfoxide (DMSO) were purchased from Sigma (St Louis, MO); 1-Hexadecyl- 2-methyl-3-(phenylmethyl)-1H-imi-dazolium iodide (NH125) was obtained from Tocris Bioscience (St. Louis, MO); the antibodies to phospho-eEF2, eEF-2, casepase-3, PARP, and LC3B, were purchased from Cell Signaling Technology (Danvers, MA); rabbit polyclonal anti-eEF2 kinase antibody was obtained from Novus Biologicals (Littleton, CO); p62 was purchased from Enzo Life Sciences (Plymouth Meeting, PA); β-actin antibody was obtained from Santa Cruz Biotechnology Inc (Santa Cruz, CA); eEF-2 kinase-siRNA and control siRNA were synthesized by Shanghai Gene-Pharma Co. (Shanghai, China); the Cell Counting Kit-8 (CCK-8) was purchased from DojinDo Molecular Technologies, Inc. (Rockville, MA); the Annexin V-FITC apoptosis detection kit and Matrigel were purchased from BD Biosciences (San Diego, CA); the Pierce BCA Protein Assay Kit was obtained from Thermo Scientific Corp (Hudson, New Hampshire); oligofectamine reagent was purchased from Invitrogen Corp (Carlsbad, CA); other Western blot reagents were obtained from Bio-Rad Laboratories (Hercules, CA).

Techniques: Expressing, CCK-8 Assay, Transfection, Control, Western Blot

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: BMC cancer

Article Title: Screening colorectal cancer associated autoantigens through multi-omics analysis and diagnostic performance evaluation of corresponding autoantibodies.

doi: 10.1186/s12885-025-14080-5

Figure Lengend Snippet: Fig. 1 The flow diagram of this study. This study was conducted in two steps: discovery of candidate TAAs (Step 1) and evaluation of TAAbs (Step 2). TAAs Tumor-associated antigens, TAAbs Tumor-associated autoantibodies, CMS Consensus Molecular Subtypes, DEG Differential Expression Analysis, ELISA Enzyme-linked immunosorbent assay, SBA Small Bowel Adenocarcinoma, SBI Specific Binding Index

Article Snippet: Recombinant proteins and the detection of TAAbs by ELISA Eight proteins (NPM1, EIF1AX, CKS1B, eEF2 (encoded by EEF2), P4HB, ANXA3, S100A11, HSP90AB1) were purchased from CUSABIO (Wuhan, China), and four proteins (HMGA1, ACTG1, maspin (encoded by SERPINB5), HKDC1) were purchased from Cloud-clone Corporation (Wuhan, China).

Techniques: Quantitative Proteomics, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Mediators of Inflammation

Article Title: Exosomes Play an Important Role in the Progression of Plasma Cell Mastitis via the PI3K-Akt-mTOR Signaling Pathway

doi: 10.1155/2019/4312016

Figure Lengend Snippet: AKT-mTOR signaling pathway activation was detected in PCM, and inhibiting exosome secretion reduced inflammation in vivo. (a) The expression of PIK3K, AKT, p-AKT, p-mTOR, HSP90AA1, and EEF2 was detected in PCM tissue sections by immunohistochemical staining. (b) Immunohistochemical staining scores were analyzed by the Mann-Whitney U test. There were no statistical differences for PIK3K and AKT in PCM tissues compared with NC tissues, while p-AKT and p-mTOR were overexpressed and HSP90AA1 and EEF2 were downexpressed in PCM tissues. ∗ p < 0.001 versus NC groups. (c) Western blots analyze changes of PI3K-Akt-mTOR pathway-related proteins. (d) Densitometric quantification for the changes of PI3K-Akt-mTOR pathway-related proteins was measured by QUANTITY ONE software. All values are expressed as the mean ± SD. ∗ p < 0.05 versus NC groups. (e, f) Pathological changes were detected by HE staining and inhibition of exosomes secretion by GW4869 significantly inhibited inflammatory cell infiltrate. Inflammatory infiltration scores were analyzed by the Mann-Whitney U test. ∗ p < 0.01.

Article Snippet: Samples were incubated with primarily indicated antibodies diluted at 1 : 100 overnight at 4°C: PI3K p85 alpha (Proteintech, 60225-1-Ig), p-AKT (Tr308) and AKT (pan) (Cell Signaling Technology), phospho-mTOR (Abcam, ab109268), HSP90AA1 (Boster, BA0369), and EEF2 (Boster, BM1733), and secondary rabbit-anti-human HistostainTM-SP Kit (SPN 9001 ZSGB-BIO) for 1 h at room temperature.

Techniques: Activation Assay, In Vivo, Expressing, Immunohistochemical staining, Staining, MANN-WHITNEY, Western Blot, Software, Inhibition