catalytic domain mutant  (Addgene inc)

Journal: The Journal of Biological Chemistry

Article Title: Genetic and environmental factors regulate the type 1 diabetes gene CTSH via differential DNA methylation

doi: 10.1016/j.jbc.2021.100774

Figure Lengend Snippet: Inhibition of Tet enzymes reduces CTSH transcription. A , relative gene expression levels of DNA methyltransferases DNMT1 (n = 9), DNMT3a (n = 11), DNMT3b (n = 7), DNA demethylases T et 1 (n = 10), T et 2 (n = 12), T et 3 (n = 10), as well as TDG (n = 6) in control islets and islets treated with IL-1β + TNF-α + IFN-γ; expression levels were normalized to β-actin and arbitrarily set to one in control islets. B , relative Tet protein activities in cytokine-treated and control human islets (n = 5). C , relative Tet protein activities in 48-h DMOG-treated and control human islets (n = 6). D , relative CTSH mRNA levels (normalized to β-actin) in 48-h DMOG-treated and control human islets (n = 6). E , relative luciferase activity of CpG21–36 luciferase plasmid when overexpressing Tet1 in HEK293 cells. Unmethylated (CpG21–36) or methylated (mCpG21–36) luciferase plasmid carrying the CTSH intron 1 CpG sites were cotransfected either with the plasmid expressing the functionally active Tet1 catalytic domain or the plasmid expressing the mutant inactive Tet1 catalytic domain at a ratio of 1:6. CTSH , cathepsin H; DMOG, dimethyloxalylglycine; HEK293, human embryonic kidney 293 cells; IFN-γ, interferon γ; IL-1β, interleukin 1β; Tet, 10–11 translocation; TNF-α, tumor necrosis factor α.

Article Snippet: For Tet1 cotransfection experiment, a plasmid expressing either the functionally active Tet1 catalytic domain (180 ng; Addgene; catalog no.: 124082) or the mutant inactive Tet1 catalytic domain (180 ng; Addgene; catalog no.: 124083) was cotransfected with either the unmethylated or the methylated pCpG-free-CTSH intron 1-lucia plasmid (30 ng), as well as the pGL4.10-firefly transfection control plasmid (100 ng).

Techniques: Inhibition, Gene Expression, Control, Expressing, Luciferase, Activity Assay, Plasmid Preparation, Methylation, Mutagenesis, Translocation Assay

Journal: Cells

Article Title: PARP10 Multi-Site Auto- and Histone MARylation Visualized by Acid-Urea Gel Electrophoresis

doi: 10.3390/cells10030654

Figure Lengend Snippet: Visualization of PARP10 819–1007 activity by polyacrylamide gel electrophoresis. ( a ) Coomassie stained SDS-PAGE, TBE-urea PAGE (TU-PAGE), and acid-urea PAGE (AU-PAGE) gels of untreated (C) and auto-MARylated PARP10. MW, molecular weight marker; ( b ) AU-PAGE gel showing PARP10 and histone H3.1 MARylation; ( c ) AU-PAGE gel showing wild type PARP10 and inactive mutant T912A, either untreated or incubated with 1 mM nicotinamide adenine dinucleotide (NAD + ); ( d ) NAD + concentration dependent mobility shift of PARP10; ( e ) PARP10 auto-MARylated using NAD + alone or in combination with the indicated NAD + analogues; ( f ) fluorescence imaging of the gel area containing 6-F-NAD + treated protein; and, ( e , f ) the positions of unmodified PARP10 are marked.

Article Snippet: Expression vectors for the PARP10 catalytic domain constructs N819-V1007 (wild type and mutants) were constructed by sub-cloning synthetic DNA fragments (GeneArt; ThermoFisher Scientific) into pNIC-28 [ ].

Techniques: Activity Assay, Polyacrylamide Gel Electrophoresis, Staining, SDS Page, Molecular Weight, Marker, Mutagenesis, Incubation, Concentration Assay, Mobility Shift, Fluorescence, Imaging

Journal: Cells

Article Title: PARP10 Multi-Site Auto- and Histone MARylation Visualized by Acid-Urea Gel Electrophoresis

doi: 10.3390/cells10030654

Figure Lengend Snippet: AU-PAGE analysis of ADP-ribosyl glycohydrolase action on auto-MARylated PARP10. PARP10 819–1007 (10 µM) was incubated sequentially with NAD + and with either TARG1 (T), MacroD2 (M), or ARH3 (A) at equimolar concentration as described to detail under Materials and Methods. Reactions were divided and processed by AU-PAGE ( upper panel ) and SDS-PAGE ( lower panel ).

Article Snippet: Expression vectors for the PARP10 catalytic domain constructs N819-V1007 (wild type and mutants) were constructed by sub-cloning synthetic DNA fragments (GeneArt; ThermoFisher Scientific) into pNIC-28 [ ].

Techniques: Incubation, Concentration Assay, SDS Page

Journal: Cells

Article Title: PARP10 Multi-Site Auto- and Histone MARylation Visualized by Acid-Urea Gel Electrophoresis

doi: 10.3390/cells10030654

Figure Lengend Snippet: Identification of PARP10 auto-MARylation sites by mass spectrometry 1 .

Article Snippet: Expression vectors for the PARP10 catalytic domain constructs N819-V1007 (wild type and mutants) were constructed by sub-cloning synthetic DNA fragments (GeneArt; ThermoFisher Scientific) into pNIC-28 [ ].

Techniques: Mass Spectrometry, Sequencing

Journal: Cells

Article Title: PARP10 Multi-Site Auto- and Histone MARylation Visualized by Acid-Urea Gel Electrophoresis

doi: 10.3390/cells10030654

Figure Lengend Snippet: AU-PAGE analysis of wild type and mutant PARP10 819–1007 activities. ( a ) Auto-MARylation; ( b ) Histone H3.1 MARylation. ( c ) Estimates of the percentages of remaining unmodified protein in the NAD + containing gel lanes (black columns, unmodified PARP10; grey columns, unmodified H3.1).

Article Snippet: Expression vectors for the PARP10 catalytic domain constructs N819-V1007 (wild type and mutants) were constructed by sub-cloning synthetic DNA fragments (GeneArt; ThermoFisher Scientific) into pNIC-28 [ ].

Techniques: Mutagenesis