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: 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