Journal: Medicine & Science in Sports & Exercise

Article Title: Preexercise Aminoacidemia and Muscle Protein Synthesis after Resistance Exercise

doi: 10.1249/mss.0b013e31825d28fa

Figure Lengend Snippet: FIGURE 5—Muscle p70 S6 kinase thr421/ser424 (A), p70 S6 kinase thr389 (B), S6 ribosomal protein (C), and eEF2 kinase (D) phosphorylation at rest and after 1 and 5 h of recovery from a bout of resistance exercise in response to a placebo feeding (PLAC) or the intake of 30-g high-quality protein fed as a bolus (BOLUS) or in a series of small feedings (PULSE). Values are mean T SEM (n = 8) presented in arbitrary units relative to >-tubulin. *, different versus rest; ‡, different versus 5 h; #, different versus BOLUS— 1 h; § different versus PLAC—1 h (P G 0.05).

Article Snippet: Polyclonal anti–phospho-mammalian target of rapamycin (mTOR) ser2448 (#2971), -p70 S6K thr421/ser424 (#9204), -eEF2K ser366 (#3691); monoclonal anti–phospho-Akt ser473 (#9271), -tuberin (TSC2) thr1462 (#3617), -S6 ribosomal protein ser235/6 (#4856), and -p70 S6K thr389 (#9206) were from Cell Signaling Technology (Danvers, MA).

Techniques: Phospho-proteomics

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: The Journal of Veterinary Medical Science

Article Title: Age-dependent increase in activity of eukaryotic elongation factor 2 kinase in mesenteric arteries from spontaneously hypertensive rats

doi: 10.1292/jvms.20-0564

Figure Lengend Snippet: Phosphorylation of eukaryotic elongation factor 2 kinase (eEF2K) at Ser500 in isolated mesenteric arteries from 4–10-week-old Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Phosphorylation of eEF2K at Ser500 was determined by Western blotting. Phosphorylation of eEF2K at Ser500 was normalized to total-eEF2K (t-eEF2K). The results were shown as fold increase relative to WKY [a (4–5-week-old): n=4, b (10-week-old): n=4]. Please note that representative t-eEF2K blots in , , and were from the same origins.

Article Snippet: Antibody sources were as follows: anti-phospho-eEF2K (p-eEF2K) Ser500 (1:200 dilution) (No. EP4451) (ECM Biosciences, Versailles, KY, USA); anti-p-eEF2K Ser366 (1:500 dilution) (No. A0071) (Assay Biotech, Sunnyvale, CA, USA); anti-total-eEF2K (t-eEF2K) (1:1,000 dilution) (No. sc-39071) (Santa Cruz Biotechnology, Dallas, TX, USA); anti-phospho-eEF2 (p-eEF2) Thr56 (1:500 dilution) (No. 905-775-100) (Assay designs, Ann Arbor, MI, USA); anti-total-eEF2 (t-eEF2) (1:500 dilution) (No. A301-688A-T) (Bethyl Laboratories, Montgomery, TX, USA); and anti-total-actin (t-actin) (1:1,000 dilution) (No. MAB1501) (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Phospho-proteomics, Isolation, Western Blot

Journal: The Journal of Veterinary Medical Science

Article Title: Age-dependent increase in activity of eukaryotic elongation factor 2 kinase in mesenteric arteries from spontaneously hypertensive rats

doi: 10.1292/jvms.20-0564

Figure Lengend Snippet: Summary of the phosphorylation and protein expression of eukaryotic elongation factor 2 (eEF2) and eEF2 kinase (eEF2K) in isolated mesenteric arteries from 4–10-week-old spontaneously hypertensive rats (SHR)

Article Snippet: Antibody sources were as follows: anti-phospho-eEF2K (p-eEF2K) Ser500 (1:200 dilution) (No. EP4451) (ECM Biosciences, Versailles, KY, USA); anti-p-eEF2K Ser366 (1:500 dilution) (No. A0071) (Assay Biotech, Sunnyvale, CA, USA); anti-total-eEF2K (t-eEF2K) (1:1,000 dilution) (No. sc-39071) (Santa Cruz Biotechnology, Dallas, TX, USA); anti-phospho-eEF2 (p-eEF2) Thr56 (1:500 dilution) (No. 905-775-100) (Assay designs, Ann Arbor, MI, USA); anti-total-eEF2 (t-eEF2) (1:500 dilution) (No. A301-688A-T) (Bethyl Laboratories, Montgomery, TX, USA); and anti-total-actin (t-actin) (1:1,000 dilution) (No. MAB1501) (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Phospho-proteomics, Expressing, Isolation

Journal: Science Advances

Article Title: Phosphorylation of shiftless is important for inhibiting the programmed −1 ribosomal frameshift

doi: 10.1126/sciadv.adw7471

Figure Lengend Snippet: ( A ) Schematic diagram of three AirID constructs used for PDB. ( B ) Working flow of the proximity biotinylation for identifying kinases responsible for SFL phosphorylation. WB, Western blot. ( C ) The expression plasmids (A) were transfected into HEK293T cells, and 0.5 μM biotins were supplemented in the cell culture to promote biotinylation. The cells were harvested and lysed 16 hours posttransfection and analyzed by Western blot. Input: proteins before SA magnetic bead enrichment. Output: proteins after SA magnetic bead enrichment. Representative of three independent experiments. ( D ) LC-MS/MS analysis of SA magnetic bead–enriched samples from three independent biological replicates. Gray dots were proteins detected in both the 10- and 35-nm groups; black dot was SFL, and red dots were Ser/Thr kinases with expression levels at least fourfold higher than the AirID group (both 10 and 35 nm), among which six kinases (indicated with blue fonts) were selected for further analysis. FC, fold change. ( E ) Left: The purple and blue circles represent Ser/Thr kinases with expression levels up-regulated at least fourfold compared to the AirID group in the 10- and 35-nm subgroups. The green circle includes 58 kinases predicted by the PhosphositePlus software. Right: Six Ser/Thr kinases were selected because they represented the consensus of three cycles. ( F ) Phos-Tag PAGE analysis of in vitro kinase assays. Six selected kinases were tested for the ability to phosphorylate SFL. Phosphorylation species are marked by red numbers. Representative of two independent experiments. ( G ) Identification of phosphorylation sites in SFL from the marked bands in (F) by LC-MS/MS. ( H ) Expression of HIV-1 proteins in HEK293T cells with knockdown of EEF2K , NEK9 , or PBK genes in the presence or absence of SFL. Representative of two independent experiments.

Article Snippet: HEK293T cells were deposited in a 10-cm dish and transfected with 15 μg of EEF2K KO plasmids (Santa Cruz Biotechnology, sc-402166), NEK9 knockout (KO) plasmids (Ubigene, YKO-RP003-hNEK9 [gRNA1], YKO-RP003-hNEK9 [gRNA2], and YKO-RP003-hNEK9 [gRNA3]), and PBK KO plasmids (Ubigene, YKO-RP003-hPBK [gRNA1], YKO-RP003-hPBK [gRNA2], and YKO-RP003-hPBK [gRNA3]) using Lipofectamine 2000, respectively.

Techniques: Construct, Phospho-proteomics, Western Blot, Expressing, Transfection, Cell Culture, Liquid Chromatography with Mass Spectroscopy, Software, In Vitro, Knockdown