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: 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

Journal: bioRxiv

Article Title: Ser500 phosphorylation acts as a conformational switch to prime eEF-2K for activation

doi: 10.1101/2025.06.30.662482

Figure Lengend Snippet: (A) The ability of recombinant wild-type eEF-2K (WT) and the phosphomimetic S500D to bind CaM was assessed via their association with a CaM labeled with the fluorophore IAEDANS (CaM IAE ) on a nondenaturing gel. The purified proteins were either co-expressed with l-phosphatase (indicated by “ λ ”; unphosphorylated at T348) or not (“ p ” indicates phosphorylated on T348). Each construct was incubated with the indicated concentrations of CaM IAE . The +Ca 2+ samples contained 150 μM free Ca 2+ . The samples were run on a native gel to separate the unbound CaM AE from the eEF-2K/CaM AE complexes. The fluorescence of the bound CaM IAE was visualized with a UV imager, and the total protein was determined by Coomassie staining. (B) An AlphaScreen assay was used to monitor competition between GST-eEF-2K (10 nM) and untagged eEF-2K mutants (0–50 nM) for His-tagged CaM (2 nM) in the presence of 1 mM Ca 2+ . (C) IC 50 values from the plot in (B) were determined for WT p (0.78 ± 0.044 nM), S500D p (0.47 ± 0.026 nM), WT λ (2.1 ± 0.14 nM), and S500D λ (0.86 ± 0.054 nM). Two replicates were used in each case with the error bars representing the standard deviation.

Article Snippet: The native proteins were transferred to a PVDF membrane for 16 h at 30V in 25 mM Tris, 192 mM glycine, 2 mM CaCl 2 , and 20% methanol and then probed with CaM (4830, Cells Signaling Technology), eEF-2K (C-12, sc-390710, Santa Cruz Biotechnology), or p T348 eEF-2K (EP4411, ECM Biosciences) specific antibodies.

Techniques: Recombinant, Labeling, Purification, Construct, Incubation, Fluorescence, Staining, Amplified Luminescent Proximity Homogenous Assay, Standard Deviation

Journal: bioRxiv

Article Title: Ser500 phosphorylation acts as a conformational switch to prime eEF-2K for activation

doi: 10.1101/2025.06.30.662482

Figure Lengend Snippet: (A) The entire gel from , showing bound and unbound CaM IAE fluorescence (512 nm excitation). The bound CaM IAE comigrates with recombinant eEF-2K (∼82 KDa) on the native gel, while the excess free CaM (∼17 KDa) migrates more rapidly through the gel. Addition of Ca 2+ causes a slight upward shift in free CaM. (B) The ability of recombinant eEF-2K proteins (“ p ” denotes presence of p T348, while “ λ ” denotes coexpression with λ-phosphatase, i.e., T348 is not phosphorylated) to bind CaM IAE on a native gel was assessed similarly to , except the incubation tubes contained 1 mM ADP and the enzyme concentration was increased from 0.67 to 2.5 µM. The CaM IAE fluorescence was measured using two different exposure times, 8 and 20 sec, to visualize low fluorescence signals. (C) Assessment of binding of recombinant eEF-2K proteins (0.2 µM) to CaM (0.2 µM) by native gel followed by western blotting with antibodies for CaM and eEF-2K. The concentrations of free Ca 2+ are indicated. (D) S500D-eEF-2K p was pre-incubated with or without purified λ-phosphatase (λ-pp) before combining with either 25 µM calcium-free CaM or 2 µM CaM with 150 µM free Ca 2+ . Samples were loaded onto a native gel and analyzed by Western blot using CaM, eEF-2K, and pT348-eEF-2K-specific antibodies.

Article Snippet: The native proteins were transferred to a PVDF membrane for 16 h at 30V in 25 mM Tris, 192 mM glycine, 2 mM CaCl 2 , and 20% methanol and then probed with CaM (4830, Cells Signaling Technology), eEF-2K (C-12, sc-390710, Santa Cruz Biotechnology), or p T348 eEF-2K (EP4411, ECM Biosciences) specific antibodies.

Techniques: Fluorescence, Recombinant, Incubation, Concentration Assay, Binding Assay, Western Blot, Purification

Journal: bioRxiv

Article Title: Ser500 phosphorylation acts as a conformational switch to prime eEF-2K for activation

doi: 10.1101/2025.06.30.662482

Figure Lengend Snippet: Schematic representation of the various eEF-2K constructs, including wild-type full-length eEF-2K, the eEF-2K TR (a 6-glycine linker has replaced missing 70 N-terminal residues, and the 359-490 segment; the linker is indicated in purple), eEF-2K ΔN (missing 70 N-terminal residues but containing an intact regulatory loop), eEF-2K Δ490-520 (a 6-glycine linker has replaced 490-520 segment), and eEF-2K Δ497-502 (a 6-glycine linker has replaced 490-502 segment). The N-terminal calmodulin-targeting motif (CTM), the α−kinase domain (KD), the regulatory loop, and the C-terminal domain (CTD) are indicated. The activating T348 and S500 sites are located at the two ends of the regulatory loop.

Article Snippet: The native proteins were transferred to a PVDF membrane for 16 h at 30V in 25 mM Tris, 192 mM glycine, 2 mM CaCl 2 , and 20% methanol and then probed with CaM (4830, Cells Signaling Technology), eEF-2K (C-12, sc-390710, Santa Cruz Biotechnology), or p T348 eEF-2K (EP4411, ECM Biosciences) specific antibodies.

Techniques: Construct

Journal: bioRxiv

Article Title: Ser500 phosphorylation acts as a conformational switch to prime eEF-2K for activation

doi: 10.1101/2025.06.30.662482

Figure Lengend Snippet: (A) Hydrogen-deuterium exchange mass spectrometry (HXMS) experiments were performed on eEF-2K ΔN alone or in a complex with CaM. T348 was dephosphorylated in all cases. The only region within eEF-2K ΔN that showed statistically significant differences between its CaM-free and CaM-bound states involved the V502-D513 segment proximal to S500. Two overlapping peptides corresponding to this region are shown. These peptides illustrate reduced 2 H incorporation in the CaM-free compared to the CaM-bound state. ( B) MCF10A eEF-2K -/- were transfected with the indicated eEF-2K mutants. eEF-2K expression and eEF-2 phosphorylation were assessed via western blotting. (C) The intrinsic activities of eEF-2K p , S500D-eEF-2K p , and eEF-2K Δ497-502 p were measured (in duplicate) using 450 μM PepS. k obs values of eEF-2K p , S500D-eEF-2K p , and eEF-2K Δ497-502 p are displayed in bar graph form. The mean values in the three cases were 0.04 s -1 , 0.9 s -1 , and 0.8 s -1 , respectively. (D) The association of recombinant eEF-2K p and corresponding mutants with the fluorescent CaM IAE via native gel.

Article Snippet: The native proteins were transferred to a PVDF membrane for 16 h at 30V in 25 mM Tris, 192 mM glycine, 2 mM CaCl 2 , and 20% methanol and then probed with CaM (4830, Cells Signaling Technology), eEF-2K (C-12, sc-390710, Santa Cruz Biotechnology), or p T348 eEF-2K (EP4411, ECM Biosciences) specific antibodies.

Techniques: Mass Spectrometry, Transfection, Expressing, Phospho-proteomics, Western Blot, Recombinant

Journal: bioRxiv

Article Title: Ser500 phosphorylation acts as a conformational switch to prime eEF-2K for activation

doi: 10.1101/2025.06.30.662482

Figure Lengend Snippet: Left panel: Wild-type eEF-2K (E, black) has low activity until bound to CaM (E•CaM). The formation of this complex (step 1) promotes autophosphorylation at T348 (step 2) to yield the active complex (E p T348 •CaM). The E p T348 form has a higher affinity for CaM than E and partially stabilizes the active complex (step 3). Right panel: Phosphorylation of S500 (mimicked by the S500D mutant, E, purple) increases the amount of active complex (E p T348 •CaM) by increasing the affinity of E for CaM (step 1), enhancing the rate of T348 autophosphorylation (step 2) and increasing the affinity of the corresponding E p T348 form for CaM (step 3). Cellular eEF-2K is phosphorylated at S500 either through CaM-induced autophosphorylation or by PKA. In the former case, phosphorylation on S500 occurs once T348 is phosphorylated and serves to trap eEF-2K in the E p T348 •CaM complex by significantly enhancing CaM affinity, i.e., affecting only step 3. PKA-mediated phosphorylation of S500, however, can influence all three steps to drive the formation of a stable E p T348 •CaM complex.

Article Snippet: The native proteins were transferred to a PVDF membrane for 16 h at 30V in 25 mM Tris, 192 mM glycine, 2 mM CaCl 2 , and 20% methanol and then probed with CaM (4830, Cells Signaling Technology), eEF-2K (C-12, sc-390710, Santa Cruz Biotechnology), or p T348 eEF-2K (EP4411, ECM Biosciences) specific antibodies.

Techniques: Activity Assay, Phospho-proteomics, Mutagenesis

Journal: Biochemistry

Article Title: Calcium/Calmodulin Stimulates the Autophosphorylation of Elongation Factor 2 Kinase on Thr-348 and Ser-500 to Regulate its Activity and Calcium Dependence

doi: 10.1021/bi201788e

Figure Lengend Snippet: (A) Characterization of anti-phospho-eEF-2K (Ser-500) antibody. The antibody was characterized against 50 ng recombinant eEF-2K by immunoblotting as described under ‘Experimental Procedures’. Lanes: 1 – untreated eEF-2K WT; 2 – autophosphorylated eEF-2K WT; 3 – untreated eEF-2K S500A; 4 – autophosphorylated eEF-2K S500A; 5 – untreated eEF-2K S500D; 6 – autophosphorylated eEF-2K S500D. (B) Time course of incorporation of phosphate at Ser-500. eEF-2K (500 nM) was allowed to autophosphorylate in the presence of 5 µM CaM and 50 µM free Ca2+. At the indicated times, 50 ng of eEF-2K were removed and the reaction quenched with hot SDS-PAGE sample loading buffer. The samples were then analyzed by Western blotting using the anti-phospho-eEF-2K (Ser-500) antibody as described under ‘Experimental Procedures’. (C) Graphical representation of (B). Western blots were quantified using ImageJ, and data then plotted as the percent phosphorylation of Ser-500 against autophosphorylation time. Inset: Expansion of the data for 0–70 min. Experiments were performed in duplicate, and error bars represent the standard deviation. (D) Buffers used are described under ‘Experimental Procedures’. Assays were performed with eEF-2K enzyme, ± 50 µM free Ca2+ and ± 2 µM calmodulin. EGTA (1 mM) was added to all assays conducted in the absence of Ca2+. For eEF-2K WT, S500A and S500D assayed in the presence of both Ca2+ and CaM, and eEF-2K S500D assayed in the presence of only CaM, activities were much higher than the basal level of kinase activity, and hence only 5 nM of kinase was used. For all the other assays, 50 nM eEF-2K was used in order to detect an increase in kinase activity over the basal level. Kinase activity was determined by measuring the rate of phosphorylation of the peptide (µM.s−1). Activities of the mutants are reported as the percentage of the wild type activity.

Article Snippet: Monitoring incorporation of phosphate at Ser-500 by immunoblotting To analyze the time course of phosphate incorporation at Ser-500, ECM Biosciences generated affinity-purified rabbit polyclonal anti-eEF-2K (Ser-500) phospho-specific antibodies, which were used in Western blotting.

Techniques: Recombinant, Western Blot, SDS Page, Standard Deviation, Activity Assay

Journal: Biochemistry

Article Title: Calcium/Calmodulin Stimulates the Autophosphorylation of Elongation Factor 2 Kinase on Thr-348 and Ser-500 to Regulate its Activity and Calcium Dependence

doi: 10.1021/bi201788e

Figure Lengend Snippet: Summary of the various phosphorylated residues on eEF-2K. Components are color coded as follows: ( - red) – suggested to be involved in the negative regulation of eEF-2K activity through an inhibitory phosphorylation (these sites include Ser-78, Ser-359, Ser-366 and Ser-396). Regulation through the mTOR pathway involves the phosphorylation of Ser-366 by p70 S6 kinase, and the phosphorylation of Ser-359 and Ser-78 by at least two additional unknown kinases (22–24). It has been postulated that the Ser-78 phosphorylation acts to hinder the binding of CaM to eEF-2K (24). The cdc2-cyclin B complex has been shown to modulate eEF-2K activity via Ser-359 in a manner that is dependent on the cell cycle as well as amino acid availability, and is perhaps controlled by mTOR (25). Regulation through the MAPK cascade occurs via the phosphorylation of Ser-366 by p90RSK1 in an ERK-dependent fashion (22). In addition, the stress-activated protein kinases p38α and p38δ inhibit eEF2K via phosphorylation on Ser-396 (23). p38δ is also known to phosphorylate eEF-2K on Ser-359 (21); ( - green) – suggested to be involved in the positive regulation of eEF-2K activity through an activating phosphorylation (these sites include Ser-398 and Ser-500). Phosphorylation of Ser-398 by the energy-supply regulator AMPK is known to activate eEF-2K (29). The cAMP-dependent PKA has also been shown to activate eEF-2K via a phosphorylation on Ser-500, and in the process imparts Ca2+-independent activity to the kinase (26–28); ( - blue) – involved in autophosphorylation of eEF-2K (these sites include Thr-348, Thr-353, Ser-445, Ser-474 and Ser-500). Of the 5 autophosphorylation sites, only Thr-348 appears to be essential for activity against its substrate. Ser-500 is an autophosphorylation site and is also known to be phosphorylated by PKA, and could be the key residue responsible for autophosphorylation-induced Ca2+-independent (CaM-dependent – this work) activity (16, 17). The role of the phosphorylation at Ser-377 by MAPKAP-K2 has not yet been determined (23).

Article Snippet: Monitoring incorporation of phosphate at Ser-500 by immunoblotting To analyze the time course of phosphate incorporation at Ser-500, ECM Biosciences generated affinity-purified rabbit polyclonal anti-eEF-2K (Ser-500) phospho-specific antibodies, which were used in Western blotting.

Techniques: Activity Assay, Binding Assay

Journal: Acta Neuropathologica Communications

Article Title: Activity of translation regulator eukaryotic elongation factor-2 kinase is increased in Parkinson disease brain and its inhibition reduces alpha synuclein toxicity

doi: 10.1186/s40478-018-0554-9

Figure Lengend Snippet: eEF2K expression and activity in PD brain. a Quantitation of p-eEF2 (T56) IHC (3,3′-Diaminobenzidine-DAB staining) in postmortem hippocampus- Hip (CA1 and CA2 fields) and midbrain- MB (SN-substantia nigra, and PAG-peri-aqueductal gray matter) sections from 3 control and 6 PD cases (Additional file : Table S1; counts from at least 6 high power fields from each control or PD section; Mann–Whitney test, * p < 0.05, *** p < 0.005; error bars indicate Mean ± S.D.). b - d eEF2K mRNA expression in control and PD striatum ( b ), medial substantia nigra ( b ) and dorsal nucleus of vagus nerve ( d ). The following publicly available transcriptomic profile datasets were analyzed on the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) platform: Striatum ( b )- dataset GEO accession # GSE28894, Illumina human Ref-8 v2.0 expression beadchip platform, probe ID ILMN_1789171, controls n = 15 and PD n = 15; Medial substantia nigra ( c )- dataset GEO accession # GSE8397, Affymetrix Human Genome U133B Array, probe ID 225546_at, controls n = 8 and PD n = 15; Dorsal nucleus of vagus ( d )- dataset GEO accession # GSE43490, Agilent-014850 Whole Human Genome Microarray, probe ID A_24_P716162, controls n = 6 and PD n = 7. (Mann–Whitney test, * p < 0.05; error bars in 3b-d indicate Mean ± S.D.). e Relative eEF2K mRNA expression in human iPSCs derived cultured midbrain control (WT, n = 3) or A53T ( n = 2) mutation carrying organoids (T-test, * p < 0.05; error bars indicate Mean ± S.D.)

Article Snippet: Additional reagents and biochemical assays employed during these studies include: pool of small interference RNAs (SiRNAs) targeting mouse eEF2K (Santa Cruz, #sc-39,012), Cell Titer Glo ATP measurement kit (Promega, #G7570), Lactate dehydrogenase (LDH) fluorometric assay (Novus Biologicals, #NBP2–54851), Seahorse Mito stress test kit (Agilent, #103015–100), 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescent ROS reagent (ThermoFisher, #D399), and MitoTracker Green fluorescent reagent for mitochondrial mass (ThermoFisher, #M7514).

Techniques: Expressing, Activity Assay, Quantitation Assay, Staining, Control, MANN-WHITNEY, Gene Expression, Microarray, Derivative Assay, Cell Culture, Mutagenesis

Journal: Acta Neuropathologica Communications

Article Title: Activity of translation regulator eukaryotic elongation factor-2 kinase is increased in Parkinson disease brain and its inhibition reduces alpha synuclein toxicity

doi: 10.1186/s40478-018-0554-9

Figure Lengend Snippet: Brain eEF2K expression and activity in transgenic M83 +/+ PD mice. a eEF2K mRNA levels in whole brain homogenates from transgenic M83 +/+ PD mice intramuscularly (IM) injected bilaterally with phosphate buffered saline (PBS, n = 10) or pre-formed fibrillar (PFF, n = 13) mouse wild type AS. (Mann–Whitney test, *** p < 0.005; error bars indicate Mean ± S.D.). b - c Western blot analysis of p-eEF2 (T56) and p-ASyn (S129) in whole brain homogenates from transgenic M83 +/+ PD mice intramuscularly (IM) injected bilaterally with phosphate buffered saline (PBS) or pre-formed fibrillar (PFF) mouse wild type AS ( b ), and corresponding densitometry analysis ( c ) ( n = 7/group; Mann–Whitney test, * p < 0.05, *** p < 0.005; error bars indicate Mean ± S.D.)

Article Snippet: Additional reagents and biochemical assays employed during these studies include: pool of small interference RNAs (SiRNAs) targeting mouse eEF2K (Santa Cruz, #sc-39,012), Cell Titer Glo ATP measurement kit (Promega, #G7570), Lactate dehydrogenase (LDH) fluorometric assay (Novus Biologicals, #NBP2–54851), Seahorse Mito stress test kit (Agilent, #103015–100), 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescent ROS reagent (ThermoFisher, #D399), and MitoTracker Green fluorescent reagent for mitochondrial mass (ThermoFisher, #M7514).

Techniques: Expressing, Activity Assay, Transgenic Assay, Injection, Saline, MANN-WHITNEY, Western Blot

Journal: Acta Neuropathologica Communications

Article Title: Activity of translation regulator eukaryotic elongation factor-2 kinase is increased in Parkinson disease brain and its inhibition reduces alpha synuclein toxicity

doi: 10.1186/s40478-018-0554-9

Figure Lengend Snippet: Effects of eEF2K inhibition on human AS cytotoxicity in differentiated N2A cells. a - b Western blot analysis of p-eEF2 (T56) levels in N2A cells subsequent to transient overexpression of human wild type or mutant A53T AS, with or without siRNA mediated eEF2K knockdown ( a ), and corresponding densitometry analysis ( b ) ( n = 6–9/group from three independent experiments; One-way ANOVA post-hoc Bonferroni test, * p < 0.05, *** p < 0.005; error bars indicate Mean ± S.E.M). c Measurements of cytotoxicity by lactate dehydrogenase-LDH release in the culture medium ( c ) and FACS analysis of propidium iodide-PI staining ( d ) in N2A cells subsequent to transient overexpression of human wild type or mutant A53T AS, with or without siRNA mediated eEF2K knockdown ( n = 9–12/group from three independent experiments; One-way ANOVA post-hoc Bonferroni test, * p < 0.05, ** p < 0.01, *** p < 0.005, NS = not significant; error bars indicate Mean ± S.D.)

Article Snippet: Additional reagents and biochemical assays employed during these studies include: pool of small interference RNAs (SiRNAs) targeting mouse eEF2K (Santa Cruz, #sc-39,012), Cell Titer Glo ATP measurement kit (Promega, #G7570), Lactate dehydrogenase (LDH) fluorometric assay (Novus Biologicals, #NBP2–54851), Seahorse Mito stress test kit (Agilent, #103015–100), 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescent ROS reagent (ThermoFisher, #D399), and MitoTracker Green fluorescent reagent for mitochondrial mass (ThermoFisher, #M7514).

Techniques: Inhibition, Western Blot, Over Expression, Mutagenesis, Knockdown, Staining

Journal: Acta Neuropathologica Communications

Article Title: Activity of translation regulator eukaryotic elongation factor-2 kinase is increased in Parkinson disease brain and its inhibition reduces alpha synuclein toxicity

doi: 10.1186/s40478-018-0554-9

Figure Lengend Snippet: Effects of eEF2K inhibition on mitochondrial dysfunction and oxidative stress induced by human AS in differentiated N2A cells. a - b Measurements of basal oxygen consumption rate-OCR ( b ) and ATP levels ( c ) in N2A cells subsequent to transient overexpression of human wild type or mutant A53T AS, with or without siRNA mediated eEF2K knockdown ( n = 9–12/group from three independent experiments; Unpaired T-test, * p < 0.05, ** p < 0.01, *** p < 0.005; error bars indicate Mean ± S.D.). c Flow cytometry analysis of reactive oxygen species (ROS), measured by DCFDA staining, in N2A cells subsequent to transient overexpression of human wild type or mutant A53T AS, with or without siRNA mediated eEF2K knockdown ( n = 9/group from three independent experiments; Unpaired T-test, * p < 0.05, ** p < 0.01, *** p < 0.005; error bars indicate Mean ± S.D.)

Article Snippet: Additional reagents and biochemical assays employed during these studies include: pool of small interference RNAs (SiRNAs) targeting mouse eEF2K (Santa Cruz, #sc-39,012), Cell Titer Glo ATP measurement kit (Promega, #G7570), Lactate dehydrogenase (LDH) fluorometric assay (Novus Biologicals, #NBP2–54851), Seahorse Mito stress test kit (Agilent, #103015–100), 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescent ROS reagent (ThermoFisher, #D399), and MitoTracker Green fluorescent reagent for mitochondrial mass (ThermoFisher, #M7514).

Techniques: Inhibition, Over Expression, Mutagenesis, Knockdown, Flow Cytometry, Staining