mouse monoclonal anti phosphorylated eukaryotic elongation factor 2 kinase p eef2k antibody  (Santa Cruz Biotechnology)

Journal: Journal of Biomedical Research

Article Title: Mouse KL2 is a unique MTSE involved in chromosome-based spindle organization and regulated by multiple kinases during female meiosis

doi: 10.7555/JBR.37.20230290

Figure Lengend Snippet: KL2 localization to the chromosomes was modulated by phosphorylated eukaryotic elongation factor-2 kinase (p-eEF2K) in mouse oocytes.

Article Snippet: Primary antibodies of mouse monoclonal anti-β-tubulin (Cat. #sc-5274, 1∶500), rabbit polyclonal anti-katanin p60 AL2 (N-15) (Cat. #sc-84855, 1∶1000),goat polyclonal anti-katanin p60 A1 (M-13) (Cat. #sc-109299, 1∶1000), mouse monoclonal anti-katanin p60 AL1 (A-10) (Cat. #sc-373814, 1∶500), rabbit polyclonal anti-fidgetin (H-146) (Cat. #sc-68343, 1∶750), goat polyclonal anti-fidgetin like 1 (FIGNL1) (C-12) (Cat. #sc-138278, 1∶1000), goat polyclonal anti-FIGNL2 (G-14) (Cat. #sc-242820, 1∶800), mouse monoclonal anti-phosphorylated eukaryotic elongation factor-2 kinase (p-eEF2K) antibody (H-2) (Cat. #sc-377536, 1∶1000), and rabbit polyclonal anti-p-aurora B (phospho T232) (Cat. #ab115793, 1∶500) antibodies were purchased from Santa Cruz (St. Louis, MO, USA).

Techniques:

Journal: Journal of Biomedical Research

Article Title: Mouse KL2 is a unique MTSE involved in chromosome-based spindle organization and regulated by multiple kinases during female meiosis

doi: 10.7555/JBR.37.20230290

Figure Lengend Snippet: Aurora B, p-eEF2K, and ERK 1/2 coordinate to modulate the chromosomal localization of KL2 in a "multilevel focus-adjusting" manner in mouse oocytes.

Article Snippet: Primary antibodies of mouse monoclonal anti-β-tubulin (Cat. #sc-5274, 1∶500), rabbit polyclonal anti-katanin p60 AL2 (N-15) (Cat. #sc-84855, 1∶1000),goat polyclonal anti-katanin p60 A1 (M-13) (Cat. #sc-109299, 1∶1000), mouse monoclonal anti-katanin p60 AL1 (A-10) (Cat. #sc-373814, 1∶500), rabbit polyclonal anti-fidgetin (H-146) (Cat. #sc-68343, 1∶750), goat polyclonal anti-fidgetin like 1 (FIGNL1) (C-12) (Cat. #sc-138278, 1∶1000), goat polyclonal anti-FIGNL2 (G-14) (Cat. #sc-242820, 1∶800), mouse monoclonal anti-phosphorylated eukaryotic elongation factor-2 kinase (p-eEF2K) antibody (H-2) (Cat. #sc-377536, 1∶1000), and rabbit polyclonal anti-p-aurora B (phospho T232) (Cat. #ab115793, 1∶500) antibodies were purchased from Santa Cruz (St. Louis, MO, USA).

Techniques:

Journal: Cell death & disease

Article Title: The eEF2 kinase coordinates the DNA damage response to cisplatin by supporting p53 activation.

doi: 10.1038/s41419-024-06891-4

Figure Lengend Snippet: Fig. 1 eEF2K mediates cellular susceptibility towards cisplatin. A Viability of Eef2k+/+ and Eef2k−/−MEFs treated with the indicated concentrations of cisplatin (CisPt) or vehicle control for 48 h as measured by an MTT assay (n = 3). Untreated condition was set to 100% for each cell line. B Viability of HEK293 cells stably expressing individual shRNAs targeting eEF2K (sh-eEF2K1 and sh-eEF2K2) or scrambled control (sh-scr) treated with the indicated concentrations of CisPt or vehicle control for 72 h as measured by an MTT assay (n = 3). Untreated condition was set to 100% for each cell line. C Cell death rates of Eef2k+/+ and Eef2k−/−MEFs treated with the indicated concentrations of CisPt or vehicle control for 48 h as indicated, as measured using trypan blue staining (n = 3). D Level of apoptosis markers in Eef2k+/+ and Eef2k−/−

Article Snippet: Pooled mouse eEF2K siRNA (sc-39011, Santa Cruz, Dallas, Texas, USA) was used to knockdown eEF2K expression. siRNAs-mediated knockdown P53 expression was performed using mouse p53 siRNAs with the following sequences si-p53-1 GUA AAC GCU UCG AGA UGU U and si-p53-2 AAA UUU GUA UCC CGA GUA U (Dharmacon, Lafayette, Colorado, USA).

Techniques: Control, MTT Assay, Stable Transfection, Expressing, Staining

Journal: Cell death & disease

Article Title: The eEF2 kinase coordinates the DNA damage response to cisplatin by supporting p53 activation.

doi: 10.1038/s41419-024-06891-4

Figure Lengend Snippet: Fig. 2 eEF2K supports the induction of the DDR pathways in response to cisplatin. A Scheme illustrating ATM/ATR signalling in response to DNA damage. B Level of activity of the ATM/ATR-dependent DDR pathways in Eef2k+/+ and Eef2k−/−MEFs treated with CisPt (5 μM) for the indicated times, as measured with immunoblot analysis using the indicated antibodies. The immunoblots are representative of three independent experiments. C Level of activity of the ATM/ATR-dependent DDR pathways in MEFs transfected with siRNA targeting eEF2K (si-Eef2k) or scrambled control (scr) treated with CisPt (5 μM) for the indicated times, as measured with immunoblot analysis using the indicated antibodies. The immunoblots are representative of three independent experiments. * indicates unspecific bands (as previously reported [32]). D Level of γH2AX foci formation in Eef2k+/+ and Eef2k−/−MEFs treated with CisPt (50 μM) or vehicle control for 2 h, as determined by immunofluorescence using a γH2AX antibody (n = 3). E Level of 53BP1 foci formation in Eef2k+/+ and Eef2k−/−MEFs treated with CisPt (50 μM) or vehicle control for 2 h, as determined by immunofluorescence using a 53BP1 antibody (n = 3). Data are expressed as mean ± SD; ***P < 0.005.

Article Snippet: Pooled mouse eEF2K siRNA (sc-39011, Santa Cruz, Dallas, Texas, USA) was used to knockdown eEF2K expression. siRNAs-mediated knockdown P53 expression was performed using mouse p53 siRNAs with the following sequences si-p53-1 GUA AAC GCU UCG AGA UGU U and si-p53-2 AAA UUU GUA UCC CGA GUA U (Dharmacon, Lafayette, Colorado, USA).

Techniques: Activity Assay, Western Blot, Transfection, Control

Journal: Cell death & disease

Article Title: The eEF2 kinase coordinates the DNA damage response to cisplatin by supporting p53 activation.

doi: 10.1038/s41419-024-06891-4

Figure Lengend Snippet: Fig. 3 eEF2K promotes enhanced DNA damage repair in response to cisplatin. A Level of DNA damage in Eef2k+/+ and Eef2k−/−MEFs treated with vehicle (-) or with CisPt (5 μM) for 2 h followed by 2 or 24 h period of recovery, as measured by modified alkali comet assay (n = 3). B Level of DNA damage in HEK293 cells stably expressing sh-eEF2K1 or sh-scr treated with vehicle (-) or with CisPt (5 μM) for 2 h followed by 2 or 24 h period of recovery, as measured by modified alkali comet assay (n = 3). C Level of γH2AX foci formation and resolution in Eef2k+/+ and Eef2k−/−MEFs treated with vehicle (-) or with CisPt (5 μM) for 2 h followed by 0 to 48 h period of recovery, as determined by immunofluorescence using a γH2AX antibody (n = 3). D Micronuclei formation in Eef2k+/+ and Eef2k−/−MEFs treated with vehicle (-) or with CisPt (5 μM) for 2 h followed by 16 or 24 h period of recovery, as measured using DAPI staining. Red arrows indicating micronuclei positive cells and grey arrows indicating micronuclei negative cells (n = 3). E Level of ERCC1 and XPF expression in Eef2k+/+ and Eef2k−/−MEFs treated with CisPt (5 μM) for the indicated times, as measured with immunoblot analysis using the indicated antibodies. Data are expressed as mean ± SD; *P < 0.05, ***P < 0.005.

Article Snippet: Pooled mouse eEF2K siRNA (sc-39011, Santa Cruz, Dallas, Texas, USA) was used to knockdown eEF2K expression. siRNAs-mediated knockdown P53 expression was performed using mouse p53 siRNAs with the following sequences si-p53-1 GUA AAC GCU UCG AGA UGU U and si-p53-2 AAA UUU GUA UCC CGA GUA U (Dharmacon, Lafayette, Colorado, USA).

Techniques: Single Cell Gel Electrophoresis, Stable Transfection, Expressing, Staining, Western Blot

Journal: Cell death & disease

Article Title: The eEF2 kinase coordinates the DNA damage response to cisplatin by supporting p53 activation.

doi: 10.1038/s41419-024-06891-4

Figure Lengend Snippet: Fig. 4 eEF2K-mediated cellular susceptibility towards cisplatin is dependent on p53 activation and expression. A Level of p53 activation in Eef2k+/+ and Eef2k−/−MEFs treated with CisPt (50 μM) for the indicated times, as measured with immunoblot analysis using the indicated antibodies. B Level of p53 activation in HEK293 cells stably expressing sh-eEF2K1, sh-eEF2K2, or sh-scr treated with CisPt (50 μM) for the indicated times, as measured with immunoblot using the indicated antibodies. C, D Viability of Eef2k+/+ MEFs (C) or Eef2k−/−MEFs (D) transfected with individual siRNA targeting p53 (si-p53-1 and si-p53-2) or scrambled control (si-scr) treated with the indicated concentrations of cisplatin (CisPt) or vehicle control for 48 h, using an MTT assay (n = 3). Data are expressed as mean ± SD; *P < 0.05, ***P < 0.005.

Article Snippet: Pooled mouse eEF2K siRNA (sc-39011, Santa Cruz, Dallas, Texas, USA) was used to knockdown eEF2K expression. siRNAs-mediated knockdown P53 expression was performed using mouse p53 siRNAs with the following sequences si-p53-1 GUA AAC GCU UCG AGA UGU U and si-p53-2 AAA UUU GUA UCC CGA GUA U (Dharmacon, Lafayette, Colorado, USA).

Techniques: Activation Assay, Expressing, Western Blot, Stable Transfection, Transfection, Control, MTT Assay

Journal: Cell death & disease

Article Title: The eEF2 kinase coordinates the DNA damage response to cisplatin by supporting p53 activation.

doi: 10.1038/s41419-024-06891-4

Figure Lengend Snippet: Fig. 5 The eEF2K ortholog, efk-1, mediates germ cell death in Caenorhabditis elegans in response to cisplatin. A Level of germ cell death in wild type (wt) and efk-1(ok3609) (efk-1) adult C. elegans treated with the indicated concentrations of CisPt for 48 h, as measured by number of germ cell corpses per gonad arm. B Egg production in wild type (wt) and efk-1 knockout (efk-1) adult C. elegans treated with the indicated concentrations of CisPt for 48 h, as measured by number of eggs produced per worm per hour. C Percentage of viable eggs produced by wt and efk-1 knockout (efk-1) adult C. elegans treated as in (B). D Proposed model for the role of eEF2K in the DDR triggered by cisplatin. Data are expressed as mean ± SD; ***P < 0.001.

Article Snippet: Pooled mouse eEF2K siRNA (sc-39011, Santa Cruz, Dallas, Texas, USA) was used to knockdown eEF2K expression. siRNAs-mediated knockdown P53 expression was performed using mouse p53 siRNAs with the following sequences si-p53-1 GUA AAC GCU UCG AGA UGU U and si-p53-2 AAA UUU GUA UCC CGA GUA U (Dharmacon, Lafayette, Colorado, USA).

Techniques: Knock-Out, Produced

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