Journal: Cell reports

Article Title: 2-Hydroxyglutarate destabilizes chromatin regulatory landscape and lineage fidelity to promote cellular heterogeneity

doi: 10.1016/j.celrep.2021.110220

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-pH2AX [S139] (Clone N1-431) , Fluidigm , Cat# 3165036D.

Techniques: Recombinant, Control, Blocking Assay, Suspension, Electron Microscopy, Purification, Whole Genome Amplification, SYBR Green Assay, Labeling, Staining, Methylation Sequencing, Methylated DNA Immunoprecipitation, Biomarker Discovery, Software, Pyromark Assay

Journal: Scientific Reports

Article Title: Pulmonary hypertension without heart failure causes cardiorenal syndrome in a porcine model

doi: 10.1038/s41598-023-36124-1

Figure Lengend Snippet: Evaluation and quantification of tubular lesions (Masson's trichrome), automatic quantization of pH2AX and Ki67 in kidney cells. ( A ): Histological section of kidney in a PH piglet, Masson's Trichrome staining (white arrow: luminal dilation and epithelial thinning). ( B ) Histological section of kidney in a SHAM piglet, Masson's Trichrome staining. ( C ) Semi-quantitative evaluation (0 to 3) of tubular lesions by a pathologist, blinded. ( D ) Automatic quantization of pH2AX in cortex and cortico-medullary junction in kidney cells (expressed in percentage of positive cells). ( E ) Automatic quantization of Ki67 in tubular kidney cells (expressed in percentage of positive cells). PH: pulmonary hypertension.

Article Snippet: Immunofluorescence staining was performed with the rabbit monoclonal antibody against pH2AX-S139 (1/200, APO 687, AB clonal, MA, USA ) overnight.

Techniques: Staining

Journal: Nature Communications

Article Title: Eya3 partners with PP2A to induce c-Myc stabilization and tumor progression

doi: 10.1038/s41467-018-03327-4

Figure Lengend Snippet: The Thr phosphatase activity of Eya3 arises from its association with PP2A. a SDS PAGE gel shows mEya3 purified from E. coli (E-Eya3) and mammalian HEK293FT cells (M-Eya3). b Thr and c Tyr phosphatase activities of E-Eya3 and M-Eya3 were analyzed in malachite-green-based phosphatase assays using phosphor-Thr (KRpTIRR) or phosphor-Tyr (pH2AX, KATQASQEpY) peptide as substrates. All phosphatase activities were normalized to the amount of protein used in the assay. The phosphatase activity of M-Eya3 was set to 100%. All control reactions (Ctrl) in Fig. 1 refer to samples in which no protein was added. d Immunoprecipitation (IP) using a Flag antibody was conducted on lysates of HEK293FT cells transfected with empty vector (EV), Flag-Eya3 (Eya3 WT ) or Flag-H79A mutant Eya3 (Eya3 H79A ). Western blot analysis of IPs shows that the H79A mutation diminishes the interaction of Eya3 with PP2A, but does not affect the interaction of Eya3 with Six1. Molecular weight markers in kilodalton are labeled on the side of western blots. e Thr and f Tyr phosphatase activities of Eya3 WT and Eya3 H79A were evaluated using Eya3 immunoprecipitated from HEK293FT cells. Eya3 WT phosphatase activity was set to 100%. g Thr phosphatase activity assay was conducted using Eya3 immunoprecipitated from scramble control (SCR), PP2A-Cα, or PP2A-Cβ KD HEK293FT cells. Left: IP using a Flag antibody followed by western blot analysis shows that similar levels of Eya3 protein were obtained from HEK293FT cells transduced with scramble shRNA, PP2A-Cα shRNA, or PP2A-Cβ shRNA, whereas Eya3 associates with less PP2A when PP2A-Cα or PP2A-Cβ is KD. Right: Thr phosphatase assay using Eya3 proteins shown in left panel. Phosphatase activity for SCR group was set to 100%. h Thr phosphatase activity assay was performed using M-Eya3 in the presence of DMSO (no inhibitor), PP2A inhibitor okadaic acid (2.5 nM), PP2B inhibitor FK506 (5 μM), or calcineurin inhibitor cyclsoporin A (5 μM). The no inhibitor group was set to 100% phosphatase activity. Data shown are mean ± SD from three independent experiments. In the phosphatase activity assay, the p -value is calculated using one-way ANOVA followed by Tukey test, ** p < 0.01, and *** p < 0.001

Article Snippet: Three hundred micrometer of pThr peptide (KRpTIRR, Milipore), pSer peptide (RRApSVA, Milipore), or pH2AX peptide (KATQASQEpY, Abgent) were incubated with 200–500 ng of Eya protein in a 25 μl phosphatase reaction.

Techniques: Activity Assay, SDS Page, Purification, Immunoprecipitation, Transfection, Plasmid Preparation, Mutagenesis, Western Blot, Molecular Weight, Labeling, Phosphatase Assay, Transduction, shRNA