cleaved dff45  (Cell Signaling Technology Inc)

Journal: International Journal of Molecular Sciences

Article Title: GSK3β Inhibition Is the Molecular Pivot That Underlies the Mir-210-Induced Attenuation of Intrinsic Apoptosis Cascade during Hypoxia

doi: 10.3390/ijms23169375

Figure Lengend Snippet: miR-210 attenuates the hypoxia-induced increase in DNA fragmentation through the inhibition of GSK3β kinase activity. ( A ) Quantitative TUNEL assay determining apoptotic DNA fragmentation, expressed as experimental blank-corrected absorbances (O.D) measured at λ 450 (450 nm). ( B ) Qualitative Western blot depicting the processing of the DFF45 into cleaved -DFF45 (p11 fragment) as a surrogate measure of caspase-3-induced DFF40 endonuclease activity. ( C ) Quantitative ELISA determining the levels of cleaved -DFF45 (p11 fragment), expressed as experimental blank-corrected absorbance (O.D) measured at λ 570 (570 nm). ( D ) Quantitative DFF40 endonuclease activity assay, expressed as experimental blank-corrected absorbances (O.D) measured at λ 405 (405 nm). miR-210 expression levels in the respective cell lysates were determined by the miR-210 hybridization immunoassay (as described in ) and are reported in . The validation of the ectopic expression of the HA-tagged ca -GSK3β-S9A mutant in the pertinent experimental groups was performed by ELISA immunoassay and is reported in ). GSK3β kinase activity (as described in ) was measured in all experimental groups to corroborate and validate the translative effects of the ectopic expression of the ca -GSK3β-S9A mutant (reported in ). All data are expressed as mean ± S.D from three technical replicates for each of the four biological replicates belonging to each experimental group ( n = 4). ** p ≤ 0.01; **** p ≤ 0.0001; ns: not significant ( p > 0.05). OE: miR-210 overexpression; O.D: optical density; S.D: standard deviation.

Article Snippet: cleaved -DFF45 (Asp 224 ) , WB 1:500 , 5 μg , Rabbit , Cell Signaling Technology , 9731 , AB_329954.

Techniques: Inhibition, Activity Assay, TUNEL Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, Hybridization, Biomarker Discovery, Mutagenesis, Over Expression, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: GSK3β Inhibition Is the Molecular Pivot That Underlies the Mir-210-Induced Attenuation of Intrinsic Apoptosis Cascade during Hypoxia

doi: 10.3390/ijms23169375

Figure Lengend Snippet: List of antibodies and antibody-blocking peptides used in the study.

Article Snippet: cleaved -DFF45 (Asp 224 ) , WB 1:500 , 5 μg , Rabbit , Cell Signaling Technology , 9731 , AB_329954.

Techniques: Enzyme-linked Immunosorbent Assay, Blocking Assay, Peptide ELISA, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: GSK3β Inhibition Is the Molecular Pivot That Underlies the Mir-210-Induced Attenuation of Intrinsic Apoptosis Cascade during Hypoxia

doi: 10.3390/ijms23169375

Figure Lengend Snippet: miR-210 attenuates the hypoxia-induced increase in DNA fragmentation through the inhibition of GSK3β kinase activity. ( A ) Quantitative TUNEL assay determining apoptotic DNA fragmentation, expressed as experimental blank-corrected absorbances (O.D) measured at λ 450 (450 nm). ( B ) Qualitative Western blot depicting the processing of the DFF45 into cleaved -DFF45 (p11 fragment) as a surrogate measure of caspase-3-induced DFF40 endonuclease activity. ( C ) Quantitative ELISA determining the levels of cleaved -DFF45 (p11 fragment), expressed as experimental blank-corrected absorbance (O.D) measured at λ 570 (570 nm). ( D ) Quantitative DFF40 endonuclease activity assay, expressed as experimental blank-corrected absorbances (O.D) measured at λ 405 (405 nm). miR-210 expression levels in the respective cell lysates were determined by the miR-210 hybridization immunoassay (as described in ) and are reported in . The validation of the ectopic expression of the HA-tagged ca -GSK3β-S9A mutant in the pertinent experimental groups was performed by ELISA immunoassay and is reported in ). GSK3β kinase activity (as described in ) was measured in all experimental groups to corroborate and validate the translative effects of the ectopic expression of the ca -GSK3β-S9A mutant (reported in ). All data are expressed as mean ± S.D from three technical replicates for each of the four biological replicates belonging to each experimental group ( n = 4). ** p ≤ 0.01; **** p ≤ 0.0001; ns: not significant ( p > 0.05). OE: miR-210 overexpression; O.D: optical density; S.D: standard deviation.

Article Snippet: cleaved -DFF45 (Asp 224 ) (biotinylated) , ELISA detection , 20 ng/well , Rabbit , Cell Signaling Technology , 9731 , AB_329954.

Techniques: Inhibition, Activity Assay, TUNEL Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, Hybridization, Biomarker Discovery, Mutagenesis, Over Expression, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: GSK3β Inhibition Is the Molecular Pivot That Underlies the Mir-210-Induced Attenuation of Intrinsic Apoptosis Cascade during Hypoxia

doi: 10.3390/ijms23169375

Figure Lengend Snippet: List of antibodies and antibody-blocking peptides used in the study.

Article Snippet: cleaved -DFF45 (Asp 224 ) (biotinylated) , ELISA detection , 20 ng/well , Rabbit , Cell Signaling Technology , 9731 , AB_329954.

Techniques: Enzyme-linked Immunosorbent Assay, Blocking Assay, Peptide ELISA, Activity Assay

Journal: Biomolecules

Article Title: EPHA3 Contributes to Epigenetic Suppression of PTEN in Radioresistant Head and Neck Cancer

doi: 10.3390/biom11040599

Figure Lengend Snippet: EPHA3 maintains PTEN suppression and Akt activation through EZH2-mediated histone methylation in radioresistant head and neck cancer cells. ( A ) Protein levels of EZH2, PTEN, pAkt, and total Akt in EPHA3 transfected HN3 cells. (-: control, +; CRISPR Activation Plasmid transfection) ( B ) Protein levels of EZH2, H3K27me, and total H3 after the silencing of EPHA3 in HN3R cells, and in EPHA3-overexpressing HN3 cells. ** p < 0.01 ( C ) Expression of PTEN, total H3, pAKT, and total Akt through suppression of H3K27me3 after treatment with the EZH2 inhibitor (2.5 µM). ( D ) ChIP assays showing endogenous EZH2 bound to the promoter region of PTEN. *** p < 0.001.

Article Snippet: Membranes were incubated with primary antibodies to EPHA3 (sc-920), DNMT1 (#5032), EZH2 (#5246), H3K27me3 (#9731), C-myc (#5605), phospho-Akt Ser473 (#4060), PTEN (#9551), and β-actin (sc-47778), all purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activation Assay, Methylation, Transfection, Control, CRISPR, Plasmid Preparation, Expressing

Journal: Biomolecules

Article Title: EPHA3 Contributes to Epigenetic Suppression of PTEN in Radioresistant Head and Neck Cancer

doi: 10.3390/biom11040599

Figure Lengend Snippet: Our hypothetical model shows the downregulation of PTEN expression in radiation-resistant cells. PTEN expression is downregulated by CpG hypermethylation in the PTEN promoter (DNMT1-mediated DNA methylation) and histone hypermethylation in H3K27me3 (EZH2-mediated histone methylation), with constitutive activation of the Akt pathway. EPHA3/c-Myc could regulate PTEN expression epigenetically by dual mechanisms.

Article Snippet: Membranes were incubated with primary antibodies to EPHA3 (sc-920), DNMT1 (#5032), EZH2 (#5246), H3K27me3 (#9731), C-myc (#5605), phospho-Akt Ser473 (#4060), PTEN (#9551), and β-actin (sc-47778), all purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, DNA Methylation Assay, Methylation, Activation Assay